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Term Paper on Plasma Membrane
Term Paper Contents:
- Term Paper on the Introduction to Plasma Membrane
- Term Paper on the Plasmalogens
- Term Paper on the Membrane Proteins
- Term Paper on the Glycoproteins
- Term Paper on the Methods for Isolation of Plasma Membrane
- Term Paper on the Molecular Structure of Plasma Membrane
- Term Paper on the Specialisation or Modifications of Plasma Membrane
- Term Paper on the Permeability of Membranes
- Term Paper on the Function of Cell Membranes in Transport
Term Paper # 1. Introduction to Plasma Membrane:
Eukaryotic cells are organised around systems of membranes. The plasma membrane envelops the cell and separates it from its surroundings; the membranes of internal organelles divide the interior into compartments with distinct biochemical environments and functions. Hydrophobic regions within membranes also provide non-aqueous environments in which certain reaction systems, such as electron transport, take place exclusively.
Realisation of the fundamental importance of membranes to cell structure developed gradually in the 1940s and 1950s. Biochemical investigations at this time revealed that some reaction systems of mitochondria and chloroplasts could proceed only if the organelles or membranous fractions of the organelle membranes were maintained in intact form. Organisation of the enzymes of these organelles on or within membranes greatly modified their catalytic properties; in some cases, dissolution of membrane structure abolished enzymatic activity completely.
These biochemical indications of the importance of membranes in cells were complemented in the late 1950s and the 1960s by the results of morphological studies with the electron microscope, which revealed the abundance of membranes in cell structure.
Interest and investigation in recent years have centered on the molecular structure and function of membranes. This research, which has involved the combined efforts of scientists in disciplines as varied as biochemistry, biophysics, physical chemistry, immunology, and cell biology, has revealed much new information about the liquid and protein components of membranes and their organisation in membrane structure.
Term Paper # 2. Plasmalogens:
Some membrane phospholipids have structures differing from the phosphor-glycerides to some degree. The plasmalogens, also called ether lipids are simile to the basic phosphor-glyceride structural plan except that the hydrocarbon chain at the 1-carbon is attached by an ether instead of an ester linkage.
The hydrocarbon chain at the 2-carbon of the backbone is attached to the usual ester linkage, and the 3-carbon carries a phosphate group and alcohol as in the phosphor-glycerides. In the plasmalogens, the alcohol most commonly found in this position ethanolamine. Plasmalogens are commonly found in nerve and muscle membranes.
The sphingolipids form a class of membrane lipids superficially similar in structure to the phosphor-glycerides. The sphingolipids differ in the chemical group forming the backbone of the molecule, which consists of a long-chain, nitrogen-containing alcohol called sphingosine.
Sphingosine contains two chemical groups that interact with other substances to form membrane sphingolipids:
i. An amino (-NH,) and
ii. A hydroxyl (- OH) group.
In membrane sphingolipids, a long-chain hydrocarbon tail derived from a fatty acid is attached by an amide linkage to the amino group. The hydroxyl group may be unbound, forming a class of membrane sphingolipids called ceramides. More commonly, the hydroxyl site is linked to a complex carbohydrate group or by a phosphate group to one of a variety of polar or charged substances, including alcohols such as choline or ethanolamine.
Figure shows sphingomyelin, a phosphate containing membrane sphingolipid of this type. The hydrocarbon chains at one end of the molecule and the polar groups at the other end also give the sphingolipids a strongly amphipathic character.
Sphingolipids with carbohydrate groups attached at the polar ends are common as the major membrane glycolipids of both plant and animal cells. They are especially abundant in plant cell membranes and in the membranes of nerve and brain cells. In mammals, practically all of the membrane glycolipids are sphingolipids.
These mammalian glycolipids are restricted almost entirely to plasma membranes, where they orient with their polar carbohydrate groups extending from the outer membrane surface. Their function in this location has been linked to cell-to-cell recognition and adhesion. Some glycolipids at the cell surface may also act as receptors that bind to specific substances taken up by cells.
Deficiencies in the metabolism of glycolipids are the cause of several important human disorders, including Tay-Sachs disease, in which normal breakdown and turnover of carbohydrate-containing sphingolipids is deficient. The sphingolipids that accumulate as a result interfere with nerve and brain functions, leading to paralysis and severe mental impairment.
The carbohydrate groups attached to membrane sphingolipids usually contain six-carbon sugars such as glucose, galactose, mannose, fucose, or one of the various forms of sialic acid. These may be linked singly or in straight or branched chains containing as many as fifteen sugar residues.
Because of the different ways in which the sugar units may combine and link together at the polar end of a sphingolipid, these molecules can have an almost endless variety of possible structures. The same sugar residues also link together to form the carbohydrate groups of membrane glycoproteins.
Sterols based on a framework of four carbon rings, are found in both plant and animal membranes. In contrast to the phospholipids and glycolipids, sterols are almost completely nonpolar substances. Polar groups are limited to a single hydroxyl at one end of the molecule. As a result, the sterols are only slightly amphipathic.
The predominant sterol of animal membranes is cholesterol. Plasma membranes in animals contain a significant quantity of cholesterol, amounting to as much as 30% of the total membrane lipids by weight. Membranes of the cell interior contain reduced amounts, ranging from as little as 2-4% of the total membrane lipids in mitochondria to 10% in membranes of the endoplasmic reticulum. Plant membranes contain cholesterol in small amounts and larger quantities of related sterols called phytosterols.
The role of cholesterol in membranes remains uncertain. The interior fluidity of membranes is altered when cholesterol is present, and cholesterol also reduces the permeability of membranes to water and other polar substances.
Whatever its functions, cholesterol is not likely to form an essential part of the basic framework of membranes since, except for plasma membranes, must cellular membranes contain relatively little cholesterol. Sterols of any kind are rare or completely absent in bacterial and blue-green algal membranes.
Term Paper # 3. Membrane Proteins:
Most membrane proteins are functional units that reflect the biological activities of membranes. Each type of cellular membrane thus has its own characteristic assemblage of proteins, in amounts that reflect the activity of the membrane in enzymatic catalysis, cell-to-cell recognition and adhesion, and transport.
Proteins isolated from most membranes separate into 20 to 30 major bands on SDS electrophoretic gels with molecular weights ranging from about 12,000 to 250,000. These major bands probably represent the lower limit in numbers of proteins actually present, because many additional proteins probably occur in amounts too small to be detected as distinct bands.
This is true of many enzymes, receptors, and transport proteins. Others co-migrate; that is, they are so similar in molecular weight that they form a single composite band. Therefore, most membranes likely contain many more species of proteins than the 20 to 30 polypeptides usually separated on gels.
One of the surprising observations about the membrane proteins isolated to date is that their content of hydrophobic amino acids is, in most cases, not significantly higher that the hydrophobic portion found in the soluble, non-membrane proteins of the cytoplasm. This was demonstrated nicely by R.A. Capaldi, who compared the relative proportions of hydrophobic and hydrophilic amino acids in several hundred cytoplasmic and membrane proteins.
Capaldi found that, with few exceptions, the insoluble membrane proteins contain approximately the same proportion of hydrophobic amino acids as the soluble cytoplasmic, non-membrane proteins-slightly less than 50%.
The hydrophobic character of membrane proteins depends instead on either their folding conformation, which places hydrophobic amino acids on the exterior and hydrophilic ones on the interior, or on segregation of hydrophobic and hydrophilic residues in different parts of the proteins’ amino acid sequence. This folding or segregation produces a protein with amphipathic properties similar to those of the membrane lipids that is, with distinctly polar and nonpolar ends or regions.
For example, in the protein glycolphorin, a major constituent of vertebrate red blood cell membranes, the middle portion of the amino acid sequence contains a stretch of more than 30 hydrophobic residues. The molecule folds so that the hydrophobic central portion of the amino acid chain is maintained as a separate, strongly hydrophobic region of the protein. Other more hydrophilic parts of the sequence also fold into separate hydrophilic domains, giving glycophorin a strongly amphipathic character.
Origins of Membrane Proteins:
Experiments tracing the incorporation of labeled amino acids have also implicated the ER in the synthesis of membrane proteins. Franke showed that within minutes of exposure to radioactive amino acids in animal cells, label could be detected membrane proteins of the rough ER. Label subsequently appeared in the Golgi complex and, after about 20 min, in the plasma membrane. Equivalent experiments by Morre with ant tissues showed the same pattern, with first incorporation label in the rough ER, followed by label of the Golgi complex and plasma membranes within 30 to 60 min.
Exposing cells to precursors of carbohydrate groups, such labeled glucose, shows that the addition of complex sugars to proteins to form glycoproteins occurs in both the ER and the Golgi complex, with greatest concentration of activity in the Golgi complex. Analysis of enzymatic activity in isolated membrane fractions also shows that the greatest concentration of enzymes attaching carbohydrate groups to proteins occurs in the Golgi complex.
These results indicate that proteins, like membrane lipids, are first incorporated into cellular membranes in the rough ER. The new membrane proteins then flow through the smooth ER and the Golgi complex to reach the plasma membrane. Presumable flow from the rough ER to the nuclear envelope also occurs.
This evidence leaves unanswered questions about how proteins with hydrophilic and hydrophobic regions are inserted into membranes at their sites of synthesis on ER ribosomes. The answers have been supplied by G. Blobel and his associates as a part of a model Blobel calls the signal hypothesis. Blobel and his associates have shown that the first series of amino acids assembled during membrane protein synthesis is primarily hydrophobic.
This produces a hydrophobic segment at the “beginning” end of the new protein. According to Blobel’s hypothesis, this segment, the signal, causes attachment of the ribosome synthesizing the protein to ER membranes. Once attached, the signal sequence, by virtue of its hydrophobic character, penetrates into the membrane. The remaining segments of the protein follow the signal into the membrane as they are assembled.
Once synthesis is complete, the final folding conformation of the new membrane protein determines its asymmetric location in the membrane bilayer through hydrophobic and hydrophilic interactions with the surrounding membrane lipids. Work by others has shown that the initial portions of the complex carbohydrate groups of membrane glycoproteins are added onto the segments of newly synthesized proteins facing the interior of the ER sacs on the side of the ER membranes opposite the ribosomes.
The carbohydrate groups are completed as the membrane segments containing the nascent glycoproteins reach the Golgi complex. The fir webbed carbohydrate units face the inside of vesicles pinching off front the Golgi. Fusion of the vesicles with the plasma membrane places the carbohydrates on the exterior surface of the cell.
About 90% of the proteins of mitochondrial and chloroplast membranes originates from the surrounding cytoplasm. There is some evidence that at least some of these proteins enter the organelles by a process analogous to the signal mechanism. In this case, the signal recognises and binds specifically to the organelles, causing insertion of the newly synthesized proteins into the organelle membranes.
The lipids and proteins are structured in membranes in the pattern proposed by the fluid mosaic model. Lipids are arranged in a fluid bilayer; proteins are suspended as globular or particulate units within the bilayer.
The proteins may be completely free to move through the fluid bilayer as individual units, or they may be fixed in position by interactions among themselves or with elements associated with the membrane such as microtubules and microfilaments. The distribution of both membrane lipids and proteins is asymmetric.
The various types of lipid molecules occur in characteristic and different proportions in the two bilayer halves of the various cellular membranes; all proteins take up specific orientations with respect to the two membrane surfaces. Both the lipid and protein components of eukaryotic cellular membranes originate in the ER and distribute by membrane flow from the ER to other regions. Lipids can also be distributed to membranes by exchange proteins.
Term Paper # 4. Glycoproteins:
Glycophorin also illustrates the typical form taken by membrane glycoproteins, the proteins with attached carbohydrate groups. One end of the glycophorin polypeptide chain carries complex sugar groups in branched and straight chains. The carbohydrate chains in glycophorin include fucose, galactose, mannose, glucosamine, and sialic acid. Other membrane glycoproteins contain straight or unbranched chains of the same and other carbohydrates in similar arrangements, in mixed groups containing from 4 to 15 residues.
In general, the same sugars occur in both membrane glycoproteins and glycolipids. The carbohydrate groups of glycoproteins and glycolipids occur almost exclusively on the outer surface of the plasma membrane, which gives the cell surface what is often described as a “sugar coaling”. More technically, the cell surface layer of carbohydrates is called the glycocalyx (the prefix glyco-is derived from the Greek glykys, meaning “sweet”).
Of the total plasma membrane lipids and proteins, usually no more than 10% by weight consists of glycolipids and glycoproteins. In animals, most of the glycocalyx is provided by glycoproteins; in plants, by glycolipids.
Among the various important functions of these substances in animals are:
a. Cell-to-cell recognition,
b. Cell adhesion, and
c. Binding specific substances taken up by cells.
Phospholipid Suspensions in Water: Bilayers:
When placed in water, phospholipids such as the phosphor-glycerides readily disperse into suspensions called bilayers. Within a bilayer, individual phospholipid molecules are packed in parallel at right angles to the plane of the bilayer, in an orientation that satisfies their amphipathic properties.
The polar phosphate groups face the surrounding aqueous medium, and the nonpolar hydrocarbon chains associate end to end in the membrane interior in a nonpolar region that excludes water.
The bilayers formed by pure phospholipid molecules suspended in water share many properties with cellular membranes. One of the most interesting is the phase transition, a rearrangement of hydrocarbon chains within bilayers brought about by changes in temperature.
At low temperatures, the hydrocarbon chains are tightly packed and their motion is restricted. This tightly packed site is termed the gel or crystalline phase. As the temperature increases, both the space separating adjacent phospholipid molecules and the motion of hydrocarbon chains increase abruptly.
At this temperature, the phospholipid bilayer “melts,” and the hydrocarbon interior becomes fluid. These changes can be followed by X-ray diffraction and other techniques, which indicate that the increased freedom of the phospholipids above the phase transition includes rotation of entire molecules around their long axis, flexing of hydrocarbon chains, and lateral movement or diffusion of individual molecules through the bilayer.
The phase transition can be precisely followed by a technique called differential calorimetry, which measures the amount of heat required to maintain a constant rate of temperature change in a bilayer. The phase transition is marked by a peak in the amount of heat absorbed by a bilayer, since the change from gel to fluid state is endothermic (absorbing rather than releasing energy).
The peak temperature for a given phospholipid depends on the length and degree of saturation of the hydrocarbon chains and the type of polar group present in the phospholipid. Generally, the longer the hydrocarbon chains, the higher the phase transition temperature. Double bonds, since they introduce “kinks” or bends in the hydrocarbon chains that interfere with tight packing, allow the bilayer to remain fluid at lower temperatures and thus reduce the melting temperature.
Measurements using differential calorimetry show that bilayers made from a single type of phospholipid undergo the phase transition at a characteristic, sharply defined temperature. Mixtures of phospholipids broaden the temperature range of the transition phase considerably.
Biological membranes undergo a similar phase transition, which can be detected by differential calorimetry as in artificial bilayers. The temperature range of such phase transitions is broad, indicating that mixtures of phospholipids rather than single types are present. The melting temperatures are such that the phospholipids of natural membranes are in the fluid state at the temperatures normally encountered by the organism.
The fluid state of bilayers has been extensively studied by several physical approaches. Among the most important of these is electron spin resonance (ESR), a technique in which a phospholipid molecule is “marked” by attachment to a chemical group containing an unpaired electron.
In the presence of an applied magnetic field, the unpaired electron in the marker group absorbs energy at certain wavelengths in the microwave range, producing a characteristic absorption spectrum with pronounced peaks. If phospholipids containing the spin marker are introduced into a membrane so that they are initially concentrated in a localised area, collisions among the molecules cause the characteristic absorption peaks to broaden.
As the marked molecules diffuse into the surrounding bilayer, collisions between them decrease and the absorption peaks narrow. From the rate at which the spectrum narrows into sharper peaks, the rate of movement or diffusion of the marked molecules through the bilayers can be determined.
From data of this type several groups of investigators have confirmed that phospholipids in gel-phase bilayers are essentially immobile and that the phospholipid molecules of bilayers at temperatures above the phase are free to move. The rate of lateral diffusion determined for individual phospholipid molecules in artificial bilayers in the fluid state is surprisingly high, about 1-2 × 10-8 cm2/s.
Measurements of ESR markers added to living membranes give equivalent results, indicating that natural membrane bilayers are in the fluid state, with diffusion rates for individual phospholipid molecules approaching the same value of 10-8 cm2/s.
Diffusion rates of this magnitude would mean that a phospholipid molecule in the plasma membrane of a bacterium could move from one end of the cell to the other in about one second. These diffusion rates apply to the lateral movement of phospholipid molecules through the plane of model bilayers and natural membranes. Movement of a molecule from one-half of a bilayer to the other, called flip-flop has different characteristics. Significantly, these characteristics differ widely between artificial bilayers, and natural membranes.
In model bilayers, flip-flop between bilayers halves is highly restricted. Estimated rates for this type of movement are so low that phospholipid molecules can be considered to be restricted essentially to their own half and to diffuse laterally on only one side. Natural membranes, in contrast, show much faster flip-flop rates, probably moderated by membrane proteins specialised for this function. The relative degree of flip-flop is fundamental to the maintenance of lipid asymmetry in membranes, a condition in which different proportions of phospholipid types are present in the two halves of a bilayer.
The Effects of Cholesterol:
Adding cholesterol to the phospholipids used to construct artificial bilayers has a marked effect on the phase transition and membrane fluidity. Addition of small amounts of cholesterol widens the temperature range over which the bilayer melts. Sufficiently large amounts (artificial bilayers will incorporate cholesterol up to a 1: 1 molar ratio with phospholipids) will eliminate the phase transition altogether.
These effects are believed to be due to the position taken by ternl in hilavers. X-ray diffraction indicates that cholesterol lines up between the phospholipid molecules of a bilayer half, with the long axis of the cholesterol molecule paralleling the hydrocarbon chains of the phospholipids. In this position, cholesterol interferes with the tight packing of hydrocarbon chains necessary for transition to the gel phase. As a result, cholesterol acts as bilayer “antifreeze” and keeps the bilayer fluid at lower temperatures.
Above the phase transition, the presence of cholesterol between the hydrocarbon chains of adjacent phospholipid molecules has the opposite effect and restricts their movement, reducing (but not eliminating) the fluidity of a bilayer at elevated temperatures. These effects are most significant for the plasma membranes of animal cells, which may contain as much as 30% cholesterol by weight, equivalent to one cholesterol molecule for every two phospholipid molecules.
Term Paper # 5. Methods for Isolation of Plasma Membrane:
The ideal isolated membrane component is that which does not have any non-membrane contamination. The isolation of the membrane can be regarded as the removal of other cell components from the cell. This operation is somewhat less difficult in the non-nucleate mammalian red blood cells.
During hemolysis the membrane structure loosen sufficiently to allow the cell contents to escape without rupturing completely. After this hemolysis the isolation of erythrocyte plasma membrane is easy. However, the product obtained depends upon the method of lysis and the pH of the liquid.
The other procedure of getting the plasma membrane is introduced by Wolport and O’Neill in 1962 by treating the Amoeba with buffered; 45% glycerol or 2.4 M sucrose solution, caused the cell contents to shrink away from the cell membrane, which retained its characteristic shape.
The membranes were fused from the cell contents by gently homogenisation and were subsequently collected in bulk by centrifuging density.
Term Paper # 6. Molecular Structure of Plasma Membrane:
All biological membranes, including the plasma membrane and the internal membranes of eukaryotic cells, have a common overall structure- they are assemblies of lipid and protein, molecules held together by non-covalent interactions. The lipid molecules are arranged as a continuous double layer 4 to 5 nm thick.
This lipid bilayer provides the basic structure of the membrane and serves as a relatively impermeable barrier to the flow of most water- soluble molecules. The protein molecules are “dissolved” in the lipid bilayer and mediate the various functions of the membrane; some serve to transport specific molecules into or out of the cell; others are enzymes that catalyse membrane-associated reactions; and still others serve as structural links between the cell’s cytoskeleton and the extracellular matrix, or as receptors for receiving and transducing chemical signals from the cell’s environment.
All cell membranes are dynamic, fluid structures most of their lipid and protein molecules are able to move about rapidly in the plane of the membrane. Membranes are also asymmetrical structures; the lipid and protein compositions of the two faces differ from one another in ways that reflect the different functions performed at the two surfaces.
Although the specific lipid and protein components vary greatly from one type of membrane to another, most of the basic structural and functional concepts are applicable to intracellular membranes as well as to plasma membranes.
After considering the structure and organisation of the main constituents of biological membranes-the lipids, proteins, and carbohydrates- we will discuss the mechanisms cells employ to transport small molecules across their plasma membranes and the very different mechanisms they use to transfer macromolecules and larger particles across this membrane.
The Lipid Bilayer:
The first indication that the lipid molecules in biological membranes are organised in a bilayer came from an experiment performed in 1925. Lipids from red blood cell membranes were extracted with acetone and floated on the surface of water. The area they occupied was then decreased by means of a movable barrier until a monomolecular film (a monolayer) was formed.
This monolayer occupied a final area about twice the surface area of the original red blood cells. Because the only membrane in a red blood cell is the plasma membrane, the experimenters concluded that the lipid molecules in this membrane must be arranged as a continuous bilayer.
The conclusion was right but it turned out to be based on two wrong assumptions that fortuitously compensated for each other. On the one hand, the acetone did not extract the entire lipid. On the other, the surface area calculated for the red blood cells was based on dried preparations and was substantially less than the true value seen in wet preparations.
Nonetheless, the conclusions drawn from this experiment had a profound influence on cell biology; as a result, the lipid bilayer became an accepted part of most models of membrane structure, long before its existence was actually established.
Harvey and Cole indicated the existence of protein by studying the surface tension of cells. This led them to propose a lipo-protein model of the cell membrane. According to this model the plasma membrane consists of two layers of lipid molecules as shown in the lipid bilayer model.
The lipid molecules have their polar regions on the outer side.
Globulin proteins are thought to be associated with the polar groups of the lipids. The non-polar hydrophobic ends of the two layers of lipids face each other, whereas their polar hydrophilic ends are associated with protein molecules by electrostatic interaction. Protein-linked polar pores are present in the membrane. These pores are formed by periodic continuity of outer and inner layers of proteins of plasma membrane.
Modifications of Danielli-Davson Membrane Model several edifications of the above arrangement have been described:
(A) Some plasma membrane has folded α-chains of proteins on both the surfaces of lipid bilayer.
(B) Coiled x-chains of helical protein on the surfaces of lipid bilayer.
(C) With globular proteins on both the surfaces.
(D) With folded proteins on both the surfaces and helical proteins extending into the pores.
(E) With folded α-chain protein on one side and globular protein on the other side.
Robertson’s Unit Membrane Model:
Unit membrane model was put forward in the year 1953 pile studying cell under electron microscope. The basic unit membrane structure was considered to be general for a wide variety of plant and animal cells. All cell organelles such as golgi body, mitochondria, endoplasmic reticulum, nuclear membrane etc. have the unit membrane structure. The unit membrane is considered to be trilaminar with a bimolecular lipid layer between two protein layers.
Two parallel outer dense osmiophilic layers of 20A° which correspond to the two protein layers. The middle light coloured osmiophobic layer is about 35A° in thickness corresponding to the hydrocarbon chains of the lipids. Thus the unit membrane is about 7A° in thickness. In this respect it resembles the Danielli-Davson model if, however differs from the Daneilli-Davson model in that the protein in asymmetrical. On the outer surface is muco-protein; while on the inner surface is non-mucoid protein.
Objections to Unit Membrane Theory:
Objections to the unit membrane model increased during the 1960s and this led to re-examinations of lipid-protein interactions and to new models. Studies of F.S. Sjostrand, of smooth endoplasmic reticular, mitochondrial and chloroplast membranes underscored the differences between observed features of membranes and the uniformity that was required by the unit membrane concept.
Mitochondrial and chloroplast membranes contain displays of particulate units in or on the membrane. The plasma membrane did not present the same mitochondrial or chloroplast membranes. It seemed that different models might be needed to describe different functional types. This unsuitable approach became unnecessary when a mosaic membrane model was proposed.
Greater Membrane Model:
Like the trilaminar model here too the lipid layer is sandwiched between two layers of structural proteins. Robertson described the different nature of outer and inner surfaces of the membrane. The inner surface was thought to be covered with unconjugated protein, and the outer surface with glycoprotein, which is superimposed on the structural protein oligosaccharide chains with negatively charged sialic acid terminals are attached to the glycoprotein.
An alternative interpretation of molecular structure of plasma membrane has been postulated by Hoffman. They have suggested that biological membranes may have a non-lamellar pattern, consisting instead of a mosaic of globular subunits known as micelles, which have a lipid core and a hydrophilic shell of polar groups.
Lipid micelles are possible building-blocks for membranes since they tend towards spontaneous association. In this model of membrane structure the protein components of the membrane may form a monolayer on either side of the plane of lipid micelles.
Individual units of the micellar mosaic might be replaced by individual enzymes molecules or by arrays of enzymes with a precise- three dimensional organisation, enabling specific functions to be ‘built in’ to the membrane structure. The spaces between the globular micelles are thought to form water-filled pores 0.4 nm (4A°) in diameter, lined partly by the polar groups of the micelles and partly by the polar groups of associate protein molecules.
Fluid Mosaic Model:
This model was proposed by Singer and Nicolson. According to this concept, the lipid molecules are arranged to form a rather continuous bilayer that forms the structural frame work of plasma membrane.
The protein molecules are arranged in two different manners. Some proteins are located exclusively adjacent to the outer and inner surfaces of lipid bilayer and are called extrinsic proteins. Other proteins penetrate lipid bilayer partially or wholly and form integral or intrinsic proteins.
The lipids and integral proteins of plasma membrane are amphipatic in nature. The term amphipatic was coined by Hartley, 1936 for those molecules which have both hydrophobic and hydrophilic groups. The amphipatic molecules tend to constitute liquid crystalline aggregates in which hydrophobic or non-polar groups are situated inside the bilayer, and hydrophilic groups are directed towards the water phase.
Therefore, the lipid molecules form a rather continuous bilayer. The integral proteins are intercalated in the lipid bilayer, with their polar regions protruding from the surface and non-polar regions embedded in the lipid bilayer.
This arrangement explains why the active sites of enzymes and antigenic glycoproteins are exposed to the outer surface of the membranes. The quasi-fluid structure of plasma membrane explains the movement of cluster of protein molecules of considerable size across the membrane.
Pores in Plasma Membrane:
Plasma membrane is perforated by pores. These have a diameter of about 0.35 nm (nanometer), slightly larger than the sodium ions. Less than 0.1 percent of the plasma membrane is perforated by pores while 99.9 percent of the cell surface is impenetrable for ions.
Several models of structures of pores have been proposed. Some of them are:
1. Structural Pores:
These are permanent cylindrical holes that interrupt the otherwise continuous bilayer sheet.
2. Dynamic Pores:
3. Paving Block Pores:
According to this concept the pores are regarded to be the corners of the closely filled nearly hexagonal paving blocks of lipid and protein subunits.
4. Protein Channel Pores:
These pores are considered to be parts of the lipid-globular protein mosaic model. These form small channels of specific proteins embedded in the membrane through which ions and small molecules can diffuse.
The ionophores are small polypeptides whose one end is hydrophobic and other hydrophilic. The hydrophobic (outer) end dissolves in the membrane while the hydrophilic end (inner side) picks up ions or water-soluble materials and dumps them on the other side. The ionophores help in exchange of substances from or into the cell.
Term Paper # 7. Specialisation or Modifications of Plasma Membrane:
With the increased resolution of the electron microscope, numerous specialisation of cell surface have been recognised.
Following description of Fawcett deals with the various specialisations of plasma membrane studied topographically:
In the intestinal epithelium microvilli are very prominent and form a compact structure that appears under the light microscope as a striated border. These microvilli, which are 0.6 to 0.8 pm long and 0.1 um in diameters, represent cytoplasmic process covered by the plasma membrane.
Within the cytoplasm core fine microfilaments are observed which in the subjacent cytoplasm form a terminal web. The outer surface of the microvilli is covered by a coat of filamentous material (fuzzy coat) composed of glycoprotein macro- molecules.
Microvilli increase the effective surface of absorption. For example, a single cell may have as many as 300 microvilli, and in a square millimeter of intestine there may be 200,000,000. The narrow spaces between the microvilli from a kind of sieve through which, substances must pass during absorption. Numerous other cells, in addition to intestinal epithelium, have microvilli, although fewer in number.
They have been found in mesothelial cells, in the epithelial cells of the gall-bladder, uterus, and yolk sac, in hepatic cells, and so forth. The brush border of the kidney tubule is similar to the striated border, although it is of larger dimensions. An amorphous substance between the microvilli gives a periodic acid Schiff reaction for polysaccharides.
Between the microvilli, at the base, the cell membrane invaginates into the apical cytoplasm. These invaginations are apparently pathways by which large quantities of flow enter by a process similar to pinocytosis.
2. Desmosomes or Macula Adherens:
Desmosomes are cell junctions found mainly in the cells of simple columnar epithelium. These occur as specilised areas along the contact surfaces. Under light microscope the desmosomes are seen as darkly stained bodies.
Under electron microscope these appear as button-like thickenings on the inner surface of plasma membranes of adjacent cells at the point of contact. The thickenings are traversed by fine cytoplasmic fibrils called tonofibrils, which form a kind of loop in a wide arc.
These filaments stabilise the junction and act as anchoring sites for the cytoplasmic structures. The plasma membranes of adjoining cells in the regions of desmosomes are separated by an intercellular space about 30-35 nm. It is filled with intervening dense coating material that forms a dark line in the middle.
It is formed of muco-polysaccharides and proteins. The desmosomes are primarily concerned with cell adhesion, but also help in maintaining cell-shape, providing it rigidity and cellular support. The former is brought about by the intercellular coating substance and later by the tonofibrils.
Sometimes, the cells are joined by bridges of cytoplasm passing between pores of cell wall or plasma membrane between the adjacent cells, such connections are called plasmodesmata. They are usually simple but anastomosing plasmodesmata may also found. Their distribution and number may also very considerably. They were discovered by Tangl and were named as such by Strasburger.
Endoplasmic reticulum often is closely associated with the cell surface, at the points where plasmodesmata are present. Through them cytoplasmic continuity is often maintained among the adjacent cells. They provide a mean for interaction between adjacent cells which are separated in other regions.
Through them, the material can pass form cell to cell. It is not known whether all plasmodesmata are similar to each other. There exists some difference because they are not only produced at the time when ceil divides but also formed spontaneously between cells that have grown into contacts with one another, e.g. tyloses in xylem vessel elements. They may occur singly or they can be aggregated into groups. In many primary walls, the plasmodesmata are usually associated with a reduced disposition of wall material, and the area then is known as primary pit or field.
These are found in the basal surface of some epithelial cells. Their structure is similar to desmosomes but these are represented by one half; their counterpart is usually represented by collagen fibrils.
5. Terminal Bars:
The terminal bars are also known as intermediary junctions or zonula adhaerens. The terminal bars are similar to desmosomes except they lack in the tonofibrils. In terminal bar the plasma membrane is thickened and the cytoplasm of thickened area is dense. The terminal bars occur in the intermediary portion of the plasma membrane of columnar cells just below the surface. The correct identity of zonula adhaerens is still questionable.
6. Membrane Interactions:
Another aspect of cell membranes that deserve discussion is the interaction between membranes of different cells. Intercellular communication is important in many cell functions and especially during development of the organism, when cells are constantly interacting with other cells.
The nature of membrane interactions may vary from complete cytoplasmic bridges between cells to localised areas of membrane junctions that may involve an area of contact as small as a few angstroms or as large as several micrometers.
The structural nature of the actual contact generally falls into one of three categories:
a. Gap junctions,
b. Tight junctions, and
c. Septate junctions.
a. Gap Junctions:
Gap junctions appear as multilayered structures when observed with the electron microscope. They appear to be two unit membranes closely opposed to each other with a 20 to 40 A° gap between. The total thickness of the entire gap junction is 170 to 190 A°, and they are found in both vertebrates and invertebrates. They are not found in skeletal muscle fibers or red blood cells.
b. Tight Junctions:
Tight junctions are found only in vertebrates and occur in cells such as epithelial cells. These junctions appear to be true fusions between the two membranes, and they are 100 to 140 A° thick.
c. Septate Junctions:
Septate junctions have been found only in invertebrates. They are much larger than the other types of junctions and are characterised by electron-dense cross bridges that extend between the two cell membranes.
Term Paper # 8. Permeability of Membranes:
The membranes of a cell pass small ions and molecules through them. The passage of ions or molecules may occur as passive diffusion, or active transport evolving the expenditure of energy.
In passive diffusion, membranes may be classed according to their degree of permeability:
A membrane of this kind allows nothing to pass through it. Certain unfertilised fish eggs, such as trout, are permeable only to gases; water labelled with deuterium does not penetrate the egg.
In this category, model membrane may be constructed to allow passage of eater molecules, but no solute particles.
(3) Selectively Permeable:
Most membranes of the cell belong to this category. Such membranes allow water and certain selected ions and small molecules to pass through, but prohibit other ions as well as small and large molecules.
(4) Dialysing Membranes:
The endothelial cells and their basement membranes of the capillaries and nephron can act as a dialyser. In this way hydrostatic pressure forces water molecules and crystalloids across the membrane down their concentration gradients while restricting the passage of colloids.
Term Paper # 9. The Function of Cell Membranes in Transport:
Substances move constantly in both directions across cellular membranes. Metabolites, including all necessary fuel substances and raw materials, enter the cell from the outside, while waste materials and cell secretions travel in the opposite direction. These movements, moderated by the plasma membrane, maintain the concentrations of molecules inside cells at the levels required for life and cellular functions.
Within eukaryotic cells additional membrane systems, such as those surrounding mitochondria, chloroplasts, and the nucleus, maintain the interior of these organelles as separate compartments with their own distinct molecular solutions. The relative rates by which ions and molecules of various kinds pass to and from the cell and its internal compartments reflect the properties of both the lipid and protein components of membranes.
Penetration through the lipid component of membranes is largely passive and proceeds in response to gradients in concentration. This passive transport is influenced by the hydrophobic, nonpolar character of the lipid component of membranes.
In general, nonpolar and hydrophobic molecules diffuse through membranes more readily than hydrophilic and polar or charged substances. Transport moderated by the protein component primarily involves movement of hydrophilic, polar, and charged molecules and ions.
In contrast to transport through the lipid part of the membranes, much of this protein-moderated movement is active; that is, it proceeds against gradients and requires the expenditure of cellular energy. The active and passive transport associated with membrane proteins has the additional characteristic of specificity- Only certain molecules and ions pass to and from cellular compartments via the protein component of membranes.
The active and passive movement of charged particles through membranes of the restriction of these particles to one side of a membrane produces electrical effects in association with the cell surfaces. Some cells, such as the nerve cells of animals or the cells of the electric organs of fish utilise and moderate these currents or charges as the basis for their specialised functions in communication or defense.
1. The Effects of Semipermeable Membranes of Diffusion:
An artificial or natural membrane placed between two regions containing particles of different chemical or electrical potential will have no effect on the final outcome if all of the molecules or ions can pass through the membrane with equal ease.
However, the net movement may be altered, sometimes in unexpected ways, if the membrane allows some of the molecules or ions to pass through much more readily than others or excludes some molecules or ions entirely. Such a membrane is said to be semipermeable.
All biological membranes have this property and thus influence the net movement caused by gradients of chemical and electrical potential. The phenomenon of osmosis provides a familiar example of the effects of introducing a semipermeable membrane between two regions containing molecules of differing chemical potential. Consider two compartments of the same volume containing water molecules and separated by a barrier that allows water molecules to pass.
If both spaces are at the same temperature and pressure, and water molecules can pass across the barrier from either side at the same rate, there will be no net movement of water between the sides. Now consider the system, in which the right compartment also contains a quantity of molecules that are larger than the water molecules and cannot pass through the barrier.
Such a system could be duplicated by separating two spaces by a cellophane barrier and placing pure water on one side and a solution of proteins in water on the other. Obviously, if the total number of molecules on either side of the semipermeable barrier is the same, the left side has fewer water molecules per unit volume than the right, because some of the total in the right side is made up of the non-diffusing, larger molecules.
Therefore, a concentration gradient can be said to exist for the water molecules in this system. More precisely, since the presence of any dissolved substance lowers the chemical potential of a solvent, the water in the right side is of lower chemical potential.
In response, a net movement of water molecules from left to right will occur, even though the initial volume, pressure, and absolute temperature of the two spaces are the same. Osmosis refers to movement of water molecules in response to a potential gradient of this type. Since osmotic movement of water occurs in response to a gradient of chemical potential, it can accomplish work. This can be demonstrated by the apparatus.
The apparatus consists of a tube containing a solution of proteins in water. The bottom of the tube is covered by a sheet of cellophane, sealed tightly to prevent leakage. The tube is suspended in a beaker of pure water. The level of the solution in the tube will rise as water molecules move from the surroundings into the tube in response to the gradient in chemical potential.
When the pressure created by the weight of the raised solution in the tube exactly balances the tendency of water molecules to move from outside to inside, the level will become stationary. At this point, the system is in equilibrium, and no further net movement of water will occur. The pressure required to leaves of the plant. On the other hand, Protista and other small cells or organisms living in fresh water must expend energy to excrete the water constantly entering by osmotic movement to keep from bursting.
Some types of cells, such as those of bacteria, blue-green algae, and almost all plants, are kept from bursting by a thick cell wall. Cells living in surroundings containing highly concentrated salt solutions have the opposite problem and must constantly expend energy to replace the water lost to the outside by osmosis.
2. The Effects of Membrane Lipids and Proteins on Passive Transport:
The cellophane film used as a semipermeable membrane in the apparatus designed to demonstrate osmosis acts essentially a uniform molecular sieve, with no chemical or physical facts beyond restricting passage of molecules beyond certain. However, the lipids and proteins of biological membranes, and chemical properties, produce a semipermeable barrier that greatly modifies the diffusion of molecules due to potential gradients.
The effects of membrane lipids and proteins in diffusion were first analysed at the turn of the century by E. Overton, who studied the behaviour of substances as they penetrated plant and animal cells. Overton observed that the major difference between diffusion across biological membranes and diffusion across artificial barriers such as cellophane is that lipid solubility modifies the rate of penetration into cells.
Generally, the more soluble a substance is in lipids, the more spindly it penetrates into living cells, up to a limit determined y molecular size. Overton’s work provided the first clue that cells are surrounded by a surface layer of lipids.
These findings were considerably expanded in 1933 by the experiments of R. Collander and H. Barlund, who compared the lipid solubility of more than 30 different substances with the amounts penetrating per unit time across the plasma membrane of cells of Chara, an alga.
Lipid solubility was evaluated by determining the lipid-water partition coefficient for each substance:
Olive oil was used as the solvent for determining lipid solubility. By plotting the permeability (amount penetrating per unit area of cell surface per unit time) against the partition coefficient for each substance, Collander and Barlund confirmed that the penetration of substances into cells is directly related to lipid solubility- the more lipid soluble, the more permeable. On this basis, they assumed that penetrating dissolve in the lipid of the plasma membrane in order to pass from outside to inside.
Later work revealed that this conclusion was an over simplification and that the proteins of membranes, unknown to Collander and Barlund, also affect permeability. When artificial membranes consisting of single phospholipid bilayers could be constructed Collander investigators and Barlund penetrate through substances studied by such membranes much more slowly than through natural membranes.
Only the most hydrophobic molecules, with relatively high partition coefficients, penetrate as fast. Few biological molecules other than the long-chain fatty acids and sterols fall into this category. Several important metabolites, including glucose, urea, glycerol, and some of the amino acids, which pass through natural membranes rapidly, penetrate artificial phospholipid bilayers only very slowly or not at all.
The differences noted in permeability were greatest for charged particles. CI– ions, for example, were found to penetrate artificial lipid films at a rate equivalent to only 1.7 × 10-18mol/ cm/s. The permeability of red blood cells to this ion is much higher, equivalent to a rate of 1.4 × 10-8 mol/cm/s.
These findings indicate that proteins modify natural membranes by increasing the solubility and rate of penetration of hydrophilic substances. Water is an interesting exception to these conclusions because it penetrates both artificial and natural membranes quite rapidly.
If assigned an arbitrary low partition coefficient, water penetrates living cells such as Chara much faster than expected, falling considerably above the essentially straight-line plot obtained for the other molecules. Penetration of water through phospholipid films takes place at a comparable rate. The basis for this exceptional behaviour is unknown.
3. Membrane Proteins and Facilitated Diffusion:
Further investigations into the rapid penetration of substances like glucose, urea, and glycerol have provided insights into the mechanisms by which membrane proteins enhance passive transport. The degree of enhancement is sometimes large. Glucose at low concentrations, for example, penetrates passively into cells about 100,000 more rapidly than expected from its partition coefficient.
Although rapid, this enhanced passive penetration still depends on diffusion. The energy required for transport is provided by a favourable gradient in a chemical potential and does not require an expenditure of cellular energy. Enhanced transport that follows gradients but proceeds at a rate significantly higher than predicted from the partition coefficient is called facilitated diffusion.
Measurement of the rate of penetration of substances transported by facilitated diffusion reveals a fundamental’ characteristic- At successively higher concentrations, the degree of enhancement drops off until at some point further increases cause no further rise in the rate of penetration. This is in sharp contrast to the behaviour of molecules that penetrate according to their partition coefficients.
For these molecules, permeability increases regularly with concentration, with no drop-off at higher levels. The drop off noted for facilitated diffusion at high concentrations closely resembles the behaviour of enzymes in catalysing biochemical reactions: As the substrate concentration increases, enzymes gradually become “saturated” and the rate of the reaction levels off.
The similarity in behaviour between the two systems indicates that facilitated diffusion is moderated by carrier molecules in natural membranes with properties similar to those of enzymes. For the same reason, the membrane carriers are assumed to combine briefly with the transported substances as a part of the facilitation mechanism. Other characteristics of the process also point to the involvement of carrier molecules resembling enzymes in facilitated diffusion.
The mechanism exhibits specificity. Each molecule transported by facilitated diffusion is carried by a separate mechanism specific only for that substance and closely related molecules. For example, the carrier system facilitating the transport of glucose will also transport the closely related sugars mannose, galactose, xylose, and arabinose. However, it will carry only the naturally occurring D isomers of these sugars and not the L isomers.
The specificity of facilitated diffusion further resembles the specificity of enzymes in that substances with structures closely related to the normally transported molecule can inhibit the rate of transport of that molecule. The similarities observed between facilitated diffusion and enzymatic catalysis indicates that the membrane sites acting on the transported molecules to increase their permeability are proteins with properties similar to those of enzymes.
Of these similarities, the specificity of facilitated diffusion provides the strongest indication that the carrier molecules are proteins, since proteins are the only known molecules that could “recognise” the transported molecules with the required degree of specificity.
4. Ionophores and the Carrier Mechanism of Facilitated Diffusion:
Further indications that facilitated diffusion depends on the activity of membrane proteins has come from research with ionophores, carried out in the laboratories of H.A. Lardy, B.C. Pressman, P. Mueller, and others. Ionophores are antibiotics that, when added to artificial phospholipid membranes, greatly increase the permeability of positively charged ions such as Na+ and K+.
Two of these ionophores, valinomycin and gramicidin, have been most studied. Both resemble protein molecules. Valinomycin consists of alternating amino and hydroxyl acids in a chain; gramicidin is formed from a chain of 15 alternating L and D amino acids. Both molecules take on a cyclic form, with hydrophobic groups exposed on exterior surfaces and oxygen’s directed toward the interior.
The interior oxygen’s line a pore or cavity extending through the inside of the molecule. When wound into its three-dimensional form, neither molecule is large enough to extend completely through the hydrophobic portion of the membrane. When placed in solution with metallic ions, the ionophores form complexes in which the ion is held in the interior of the ionophore through interactions with the oxygens lining the internal cavity.
The ion is then effectively surrounded by the ionophore. The entire outer surface of the complex is hydrophobic in character because of the nonpolar groups directed toward the surface of the ionophore. Supposedly, the ionophore forms its complex with the transported ion at the membrane surface, removing it from the surrounding aqueous medium. The ionophore then transports the ion through the hydrophobic membrane interior and releases it to the hydrophilic medium outside the membrane.
Exactly how ionophores accomplish the transfer is unclear, but one or both of two mechanisms are considered possible. In one, the ionophore acts as a mobile carrier, shuttling back and forth between the two membrane surfaces with its enclosed ion.
In the other, several ionophores stack together, forming a continuous pore or channel that extends entirely through the membrane. Experiments indicate that either mechanism may operate, depending on the ionophore. If phospholipid membranes are “frozen” by reducing their temperature below the phase transition, the activity of valinomycin in increasing the permeability of ions is greatly inhibited or eliminated.
The activity of gramicidin is unaffected by this treatment. This suggests that valinomycin is a mobile carrier and gramicidin a pore or channel former, because reducing the fluidity of the membrane interior should inhibit movement of a mobile carrier but not affect the structure or function of a pore.
Information from other sources supports these conclusions. Valinomycin can increase movement of ions across an interface between toluene and butanol that measures on the order of micrometers. It is difficult to imagine how this process could occur by any other means than shuttling by the ionophore.
Other experiments support the channel-forming activity of gramicidin. S.B. Hladky and D.A. Haydon followed changes in electrical current between two solutions containing K+ ions exposed to a voltage difference and separated by an artificial membrane containing gramicidin (current is a measure of the number of ions moving from one solution to the other).
The current in this situation was found to fluctuate in jumps, moving rapidly from one value to another in stepwise fashion. The jumps occurred much more rapidly than a molecule the size of gramicidin could move completely from one membrane surface to the other. This suggested a mechanism involving movement of a gramicidin molecule the 1 nm or so required to complete or interrupt a stack rather than diffusion of the ionophore across the entire width of the membrane.
The proteins carrying out facilitated diffusion in natural membranes may also act by one of the same mechanisms, as either mobile carriers or channel formers. Of the two possibilities, channel formation seems more compatible with the Singer Nicolson fluid mosaic model for membrane structure. According to the model, all of the integral membrane proteins have polar and nonpolar surface regions anchoring them in the membrane.
For a membrane protein to act as a mobile carrier, a polar region directed toward one surface would have to submerge in the nonpolar membrane interior to reach the other side. This would be true whether the membrane protein facilitates diffusion by shuttling from one surface to the other or by rotating or tumbling on its axis.
In his consideration of the thermodynamic implications of the fluid mosaic model, Singer has calculated that the energy required to submerge polar groups in the nonpolar membrane interior to facilitate diffusion would be prohibitively high, much higher than the amounts available from gradients in chemical or electrical potential. Channel formation, on the other hand, can easily be reconciled with the structure of membrane proteins since most are large enough to extend entirely across the membrane.
The limited experimental evidence available on membrane transport proteins supports these arguments against mobile carriers. There is evidence that the movements in membrane proteins during transport are so small that they probably involve changes in position of restricted segments of the proteins rather than rotation or diffusion of entire molecules. This was demonstrated by recent experiments in Singer’s laboratory, in which an antibody molecule was attached to a portion of a transporter protein exposed at the membrane surface.
Attachment of the antibody would be expected to prevent or greatly inhibit any overall rotation or shutting movement of the transporter protein, since any such movement would force the attached antibody protein through the membrane. In spite of the attached antibody, no inhibition of transport was noted, indicating that the protein remains essentially fixed in position in the membrane.
These results effectively rule out rotation or movement of entire transporter proteins between the membrane surfaces. Thus, channel or pore formation remains as the likely mechanism for facilitated diffusion. In this model, the protein, this extends entirely across the membrane, remains fixed in its orientation toward the polar and nonpolar regions of the membrane.
The folding of the protein’s amino acid chain may create a pore or channel through the interior of the molecule, or two or more protein molecules may align side by side to create a channel between them. During transport, a polar “active site” located in the channel and directed toward the membrane surface combines with the transported substance.
Combination causes a conformational change in this limited portion of the protein molecule, causing translation of the active site through the channel to the other side of the membrane. This conformation reduces the affinity of the active site for the transported substance, and it is released. Upon release of the substance, the protein molecule reverts to its original conformation, with the active site exposed in a position to combine with another molecule.
Calculations of the energy required for this movement are within the amounts available from commonly observed gradients in chemical or electrical potential. The mechanisms of passive transport, involving simple and facilitated diffusion, account for the movement of a wide variety of substances to and from cells.
By these mechanisms, cells are able to absorb many of the hydrophobic and hydrophilic molecules required for their metabolic reactions and to release waste materials or secreted products to the outside. These transport processes, since they are driven by potential gradients, require no added energy to proceed.
Cells of all types share a common problem in having to choose from among the many molecules and ions present in their environment exactly those substances needed to maintain life. These substances, to the exclusion of others, must be taken up by cells at rates fast enough to maintain growth, reproduction, or specialised function. For example in the kidneys of animals blood is filtered so that virtually all substances below about 30,000 daltons pass into the urine.
Some of these are:
sugar + PEP → sugar — P + pyruvate
Standard stage free energy change for this reaction is extremely favourable, about- 40 kl/mole, because of the properties of PEP outlined. The needed PEP can come directly from glycolysis or it can be regenerated from pyruvate by an enzyme unique to bacteria and plants called phosphoenolpyruvate synthetase.
The Regeneration Reaction:
pyruvate + Pi + ATP → PEP + AMP + PPi
is followed by PP, hydrolysis to pull the equilibrium to the right.
Group translocation can be broken down into two steps:
HPr is a small, 9500-dalton protein, a histidine of which gets phosphorylated PEP. The first reaction is catalysed by the cytoplasmic protein; Enzyme I. Transfer of phosphate from HP, to a sugar is catalysed by Enzyme If, which is a complex, membrane-bound protein.
Mutant cells lacking HPr or Enzyme I fail to transport actively any of the sugars handled by this system, but Enzyme II mutations typically affect the uptake of only one sugar. Hence, Enzyme II appears to be a family of proteins, each specific for a different sugar-one for glucose, one for fructose, and so on.
In fact, there is reason to believe that Enzyme II is the carrier protein itself. To understand the mechanism of group translocation or any other transport system, we should know something about the transport proteins involved. Fortunately, some progress in this task has been made, examples of which follow.
Properties of the Carriers:
To better understand the mechanism of carrier-mediated transport, we should like to be able to examine the responsible carriers in vitro, and to use them to reconstruct model transport systems.
The Isolation of Transport Proteins:
A number of molecules that apparently serve as transport proteins have now been isolated, beginning with the galactoside carrier in the mid-1960s. One of the first successes was achieved by C.F. Fox and E.P. Kennedy, who took advantage of the fact that, in the absence of galactoside, E. coli does not manufacture any significant quantity of the three proteins required specifically and solely for lactose metabolism. When a galactoside is added to the culture, however, synthesis of the required proteins begins again.
Galactoside permease, the presumed galactoside carrier, is one of the proteins. Its synthesis is inducible- i.e., it is made only in response to need. Inducibility of the galactoside transport system permitted Fox and Kennedy to look for proteins that were present in induced cultures but absent in un-induced cultures.
In addition to the other products of the lac operon, they were thus able to identify and purify a membrane associated protein, which they called the M protein that may be the sought-after carrier.
The M protein was present at about 10,000 copies per cell in the induced state, had a molecular weight of around 30,000 daltons, and in the original cells was apparently exposed to the outside surface as judged by its ability to react with externally applied reagents.
Since then, a number of other proteins have been isolated that probably serve as carriers for membrane transport, but it is difficult to prove that they actually have this function in vivo because the appropriate assay (measure of biological activity) does not exist in vitro.
One can measure their capacity to bind the substance to be transported, but of course a protein does not have to be a membrane carrier to have a specific affinity for a small molecule. Any enzyme, and a host of other proteins also fit that description.
However, some of these isolated proteins have been shown to be necessary for transport, even if it should turn out that they themselves are not carriers. For example, several proteins have been isolated from osmotically shocked bacterial cells.
When bacteria are plasmolysed in sucrose and then quickly diluted with water, their rapid re-expansion causes some protein to be released and some transport capacity to be lost.
That some of the released proteins participate in transport was inferred from the observations that:
(1) Mutant cells lacking the capacity to transport a particular substance (cryptic mutants) also fail to release the corresponding binding protein when osmotically shocked;
(2) In cases where; transport can be, induced, un-induced cells fail to release the binding protein;
(3) The binding constants between the free proteins and transportable substances have been shown to be virtually identical with the binding constants for transport of these substances in normal cells and
(4) There are reports that incubation of the shocked cells with solutions of some of the released binding proteins restores at least part of a lost transport capacity.
This group of osmotically released proteins includes individual species capable of binding calcium, sulfate, phosphate various amino acids, galactose, and other substances. They are sometimes called periplasmic binding proteins because they appear to reside in the periplasmic space between the cell membrane and the outer membrane that is part of the cell wall in gram-negative bacteria.
Their function is apparently not transport as such, but to present substrates to the true carriers. Presumably, the hydrophobic nature of the true carriers prevents them from being so easily removed from the cell. Why bacteria have evolved this extra layer of complexity in so many of their transport systems is not certain, but it probably has to do with the difficulty of getting material through their protective but complex cell walls.
There have also been a number of successes in isolating and characterising intrinsic membrane proteins that apparently are true carries. The properties of the Na+/K+-activated ATPase. The function of this protein has been proved by incorporating it into artificial membranes and demonstrating ATP- dependent ion translocation.
Similar results have been obtained with Ca2+– activated ATPase from the endoplasmic reticulum of muscle, with a H+– translocating ATPase from mitochondria, and so on. It is only a matter of time, therefore, before the mechanism of translocation is understood.
The Advantages of Protein Carriers:
From the foregoing observations and arguments, we must conclude that carrier-mediated transport across cell membranes exists and that at least some of the carriers involved are proteins. One might, however, wonder why proteins should be chosen for this role, since they are relatively large and seemingly “expensive” to make.
There are, of course, very good reasons for that choice:
(1) Proteins are specific in their capacity to bind other molecules. They may be given exactly the right shape, with precisely the right distribution of positive and negative charges, of hydrophobic and hydrophilic regions, and of reactive group of various sorts to allow them to make fine distinctions between those molecules that will be bound and those that will not.
This specificity can be of great importance to the cell, since a close regulation of metabolic efficiency can only be achieved if the entry and exist of molecules are effectively controlled. Thus, it is possible for the cell to provide a mechanism for a particular molecule to get in and out while in the presence of much larger quantities of other kinds of molecules. Furthermore, the rate of transport is easily controlled by regulating the number of specific carriers irrespective of the rate of transport of other molecules gaining entry by their own mechanisms.
(2) The capacity of proteins to alter their binding affinity for substrate has some important applications when this function is present in a transport molecule. In Na+-coupled transport, for instance, we have a requirement for co-operativity; specifically, affinity of the carrier for glucose at its “active” site is dependent on the presence of sodium ion at its “regulatory” site.
(3) Proteins, as catalysts, also make active transport possible by allowing an obligate coupling between two reaction stergetically unfavourable transports with an energetically favourable reaction such as the hydrolysis of ATP. The sodium pump, for example, depends on this kind of coupling.
And finally, it should be pointed out that the use of proteins for transport is actually a conservative choice, for they are the direct products of genes. When small organic molecules are chosen as carriers, their synthesis is apt to require not just one, but several proteins in the form of enzymes.
That is, the production of a protein carrier may require only one gene, whereas establishing a biosynthetic pathway to produce any other kind of carrier might require several enzymes, and therefore several genes.
The suitability and economy of using proteins as carriers does not preclude the use of other organic molecules, for we may easily imagine transport tasks for which proteins are no ideal. Only a few small molecular weight carriers have been isolated. This is not surprising, as they would be present in only tiny quantities in the cell and would therefore be difficult to identify. Much of the work on small organic transport molecules has centered on the ionophorous (non-carrying) antibiotics.
These are generally macro-cyclic (ring like) compounds, produced by microorganisms and capable of sequestering inorganic ions. By virtue of its lipid solubility, the ion-antibiotic complex can diffuse through a lipid barrier that would be quite impermeable to the ion itself. One of the more widely studied ionophorous antibiotics is valinomycin. It is a 36- atom ring of alternating hydroxy and amino acids, consisting of the sequence [D-valine-D-hydroxyisovalerate-L-valine-L-lactate] repeated three times.
The ionophorous antibiotics are capable of increasing the permeability of both natural and artificial lipid membranes to small inorganic ions. They often exhibit remarkable selectivity in this role. For instance, valinomycin has about a 10,000 to 1 preference for K+ over Na+, an observation that is rot easily explained on the basis of ionic size.
The biological action of the ionophorous antibiotics has been studied with model systems such as simple chloroform barriers in which ordinary salts (e.g., KCl) are quite insoluble. In one such experiment, chloroform was placed in the bottom of a U-tube with a KCl solution above it in one arm and water above it in the other.
Valinomycin, which is capable of transporting a cation-anion pair, was added to the chloroform. Although potassium is the cation favoured by valinomycin, K+Cl is not a suitable ion pair for the antibiotic. However, picrate, PC or 2, 4, 6-trinitrophenol, was found to be transportable with potassium. Thus, when potassium picrate was added to KCl solution on one side, K+Pc– ion pairs were transported across the chloroform by diffusion and concentrated on the other side until a Donnan equilibrium was reached.
[K+]l [Pc–] l = [K+]r [Pc–]r
Here Pc is the picrate anion, and L and R stand for the left and right arms of the U-tube, respectively. This equilibrium relationship was obeyed regardless of the starting concentrations of potassium picrate and potassium chloride on the two sides.
The CI– is not picked up by valinomycin and is thereby effectively ignored when the equilibrium is established. Because valinomycin itself is uncharged, it must move both a cation and an anion in order to maintain neutrality. (Charged species, of course, are generally less soluble in lipids.) Other ionophorous antibiotics are charged and may therefore transport a single ion, promoting an ion exchange (e.g., Na+ for K+, H+ for K+, etc.) or creating a voltage gradient, in which case the equilibrium position will follow the Nernst equation.
The principle is the same, however: the antibiotic alone, or the complex of antibiotic and ion (s), is soluble and can therefore diffuse across the lipid barrier. An ion or ion pair is picked up on one side, according to the equilibrium constant between it and the antibiotic, and dropped on the other side according to the equilibrium prevailing there.
Most of the ionophorous antibiotics are products secreted by microorganisms to protect themselves against order microorganisms. They kill by disrupting normal, essential ionic gradients. There are also a few non-protein carriers that may be secreted but are designed for transport within the cell from which they arise. The most closely studied is a family of bacterial iron-binding substances known collectively as siderochromes or siderophores.
They are typically secreted from the cell, bind iron, and in that state diffuse back across the bacterial membrane. This may seem inefficient, since obviously many of the carrier molecules will be lost, but in fact bacteria have an absolute requirement for iron, which is typically scarce and for which there is competition from the host. In vertebrates, for instance, iron in the blood is transported tightly bound to a protein called transferrin and therefore is not readily available.
Thus, the bacteria are fighting for survival and produce siderochromes at a rate proportional to their need.
All indications are that membranes are rapidly assembled and disassembled in cells. Measurements show that the half-life of membrane lipids and proteins is a matter of 15 to about W hours for phospholipids and 50 to 100 or so hours for proteins. Observations of the behaviour of living cells also indicate that membranes are dynamic structures that can be rapidly assembled and disassembled.
For example, one investigator has calculated that in the growing cells of a fungus, new membrane is added at the rate of 32 µm2 per cell per minute. In spherical cells doubling in size during inter phase growth, new plasma membrane surface area increases approximately 1.6 times.
Where and how are new membranes synthesized? Answering this question, an old one in cell biology, has been complicated by revelations about membranes arising from the fluid mosaic model and its supporting research. In particular the fact that membrane proteins have strongly hydrophobic and hydrophilic regions raises questions about how and where these proteins are synthesized and how they get into membranes.
How do the hydrophilic portions of proteins that have ends exposed on both sides of a membrane pass through the hydrophobic interior? How do proteins move from their point of insertion in membranes to more distant locations in the cell? Essentially the same questions apply to membrane lipids. Where are they synthesized, and how are they transported within membranes? Recent work has supplied at least partial answers to these questions.
Evidence on the Origins of Membrane Lipids:
Using radioactively labeled precursors of membrane lipids, D.J. Morre showed that in both plant and animal cells, the first uptake and incorporation of label occurs within minutes in the endoplasmic reticulum (ER). Label then appears in the Golgi complex and lastly in the plasma membrane. These observations suggest that membrane lipids are synthesized in the ER and are first incorporated into membrane bilayers in this location. The newly synthesized lipids then move through the Golgi complex to the plasma membrane.
This conclusion is supported by electron microscopy, which shows that membrane segments “flow” from the ER to the Golgi complex and then to the plasma membrane through the medium of small vesicles that pinch off from one membrane type to fuse with the next. Flow from the rough ER to the nuclear envelope is also possible, since connections between these membrane systems are also frequently seen. The fluid nature of the membrane bilayer probably provides the basis for this lipid flow through the cytoplasmic membrane system.
Localisation of the enzymes required for membrane lipid biosynthesis also supports the conclusion that initial synthesis and incorporation take place in the ER. Much of this work has been accomplished by breaking cells open and separating membranes into fractions by centrifugation. In membranes isolated in this way, the enzymes required for the terminal steps of phospholipid synthesis prove to be associated with the ER fraction.
Enzymes catalysing the final steps of cholesterol synthesis can also be identified in the ER. Membrane flow along the ER-Golgi complex-plasma membrane or ER-nuclear envelope routes readily explains how lipids, newly synthesized in the ER, are transported between these membranes.
However, it does not explain how the membranes of mitochondria and chloroplasts, which rarely show connections of any kind to other cellular membranes, receive their lipids. These organelles contain enzymes only for synthesis of a few minor membrane lipids; most of their membrane lipids apparently originate from the surrounding cytoplasm.
A series of experiments by K.W.A. Wirtz and D.B. Zilbersmit and others has shown that lipid flow between the ER and totally disconnected organelles such as mitochondria is probably promoted by soluble cytoplasmic proteins called exchange proteins. Wirtz and Zilbersmit found that mitochondria took up label very rapidly when cells were exposed to labeled lipid precursors, indicating that the lipids newly synthesized in the ER are rapidly transferred to mitochondria even though direct membranous connections cannot be detected between the two membrane systems.
Subsequently, Wirtz and Zilbersmit isolated a group of soluble cytoplasmic proteins, the exchange proteins that can promote transfer of lipid molecules between isolated ER and mitochondria and, within mitochondria, between the inner and outer mitochondrial membranes.
Others have also demonstrated exchange proteins that stimulate movement of lipids between the ER and the plasma membrane. These proteins evidently provide the means for acid transfer between the ER, mitochondria, and chloroplasts also a mechanism for lipid transfer between the ER, Golgi complex, plasma membrane, and nuclear envelope systems.
The work accomplished to date indicates that the exchange proteins can transfer lipid molecules only to the bilayer half by face. Subsequent transfer to the opposite bilayer half by flip-flop is probably moderated, by proteins forming an integral part of the membrane.