Biochemistry: The Chemical Reactions of Living Cells, Volume 1 - D. Metzler 1980

Membranes and Cell Walls
Membrane Structure
Membrane Proteins

Because Membrane Proteins are readily denatured, for a long time they resisted isolation and characterization. These challenges were eventually overcome through the adoption of radically new approaches. It turned out that A number of Proteins can be solubilized using detergents. For instance, rhodopsin—the light-sensitive pigment and primary protein component of the outer segments of retinal rod Cells—can be obtained in a solubilized form in which it undergoes normal light bleaching (Chap. 13, Sec. E). Several Enzymes have been extracted from membranes and purified via fractionation in organic Solvents, such as methanol. Membrane proteins are typically insoluble in Water. Nevertheless, Erythrocyte membranes were rendered almost completely water-soluble by Treatment with the chelating agent EDTA at a concentration of 5∙10^-3 M (Table 4-2) or with 0.1 M tetramethylammonium bromide [27]. The results of these experiments indicate that ionic interactions between proteins (or between proteins and Phospholipids) play a crucial role in maintaining membrane stability.

Polyacrylamide gel Electrophoresis of Cell/33.html">Plasma Membrane proteins in the presence of sodium dodecyl sulfate (Chap. 2, Sec. 3.6) yields 1 to 6 prominent bands alongside at least 35 less intense bands corresponding to molecular weights ranging from 10,000 to 360,000 [28]. However, certain vital membrane proteins—such as the (Na⁺ + K⁺)-dependent ATPase (Sec. B.2.c)—are present in such minute quantities (a single erythrocyte contains only a few hundred molecules of it [3, 3a]) that they cannot be identified on electrophoretograms. Mitochondrial membranes can exhibit an even more complex composition than Plasma Membranes, whereas the composition of myelin is somewhat simpler.

Roughly one-third of the protein in The erythrocyte membrane is accounted for by two hydrophobic proteins known as spectrin (or tectin). Their molecular weight ranges from 220,000 to 250,000, and together with a lower-molecular-weight protein (molecular weight 43,000), they form rod-like structures measuring approximately 3 x 200 nm. An erythrocyte contains about 2.2∙10⁵ such rods, and if placed end-to-end, they would completely cover the internal surface of the erythrocyte (130 µm²). Because spectrins fail to undergo labeling following treatment with various chemical Reagents [28] that modify amino acid residues located on the outer membrane surface (e.g., lactoperoxidase-catalyzed iodination, Chap. 10, Sec. B.6), they are believed to reside on the inner leaflet of the membrane [3, 29]. The hypothesis that these proteins are homologous to Myosin still awaits definitive proof, and their exact role in membrane function remains unclear.

Another major component of the erythrocyte membrane is a glycoprotein with a Molecular Weight of ~100,000 that is firmly anchored within the membrane and features carbohydrate chains projecting into the surrounding medium [29]. Freeze-fracture Cytology/cytology/93.html">ELECTRON MICROGRAPHS OF the membrane bilayer surfaces (Fig. 5-1, D) reveal approximately 4,200 particles with a diameter of 8 nm randomly distributed per 1 µm² of membrane area. It is quite possible that these particles correspond to the glycoprotein in question. The physiological significance of this protein remains elusive. However, it has been proposed that its molecules contain "channels" required for The transport of water [29a] and anions [29b] (bicarbonate-chloride exchange across erythrocyte membranes occurs very rapidly and is intimately linked to The Role of Hemoglobin in Oxygen transport). That this glycoprotein spans the membrane is evidenced by the fact that treating intact erythrocytes with Trypsin results in the partial proteolysis of the glycoprotein without disrupting the membrane. Furthermore, a variety of chemical modifications selectively target this protein while leaving the bulk of membrane proteins intact [3, 3a].

Of particular interest is another erythrocyte glycoprotein known as Glycophorin (sometimes designated as PAS-1) [30, 31]. It has a molecular weight of ~31,000 and is likewise embedded in the membrane. Remarkably, CARBOHYDRATES account for about 60% of its total weight. The protein moiety of glycophorin, whose Primary Structure is known, consists of a single peptide chain containing approximately 131 amino acid residues [31]. The 50-residue N-terminal fragment is exceptionally rich in Threonine and Serine. It is thought that the N-terminal segment—to which about 16 branched oligosaccharide chains are attached—projects out from The Cell membrane; in total, a single polypeptide chain bears ~160 sugar residues (Fig. 5-2). Owing to its high sialic acid content (Fig. 2-14), the glycoprotein carries a substantial negative charge. The glycophorin molecule comprises three distinct domains. Adjacent to the carbohydrate-bearing N-terminal region is a stretch of 32 hydrophobic residues that presumably forms an α-helical segment spanning The Lipid Bilayer. The third domain, located at the C-terminus, is hydrophilic and enriched in Proline, glutamic acid, and aspartic acid residues; it likely extends into the Cytoplasm (Fig. 5-2), where it can bind Calcium Ions or interact with the —NH⁺₃ groups of phospholipid HEAD groups.

If all the carbohydrate residues of glycophorin were evenly distributed across the cell surface, they could cover roughly 1/5 of the total area, forming a sparse lattice. In reality, however, their distribution appears to be non-uniform, forming localized clusters that protrude from the membrane. These carbohydrate projections have been shown to carry MN Blood Group Antigens (Sec. B.1) and a variety of other immunological determinants. They also serve as receptors for Influenza Viruses and attachment sites for plant agglutinins (Sec. B.3).

Twenty or perhaps even more Glycoproteins are embedded in the erythrocyte membrane [28b]; these are commonly referred to as intrinsic or integral proteins. Proteins that are bound less tightly to the membrane and reside primarily on its inner surface are termed peripheral proteins [32].

Myelin contains a simple yet remarkably interesting set of proteins. Approximately 30% of the total membrane protein is represented by the strongly basic A-1 protein [33], which has a molecular weight of ~18,000. The complete Amino Acid Sequence of human A-1 protein has been determined. It is believed to adopt an extended ß-conformation, with four closely spaced proline residues near the center of the polypeptide chain allowing it to form a hairpin loop. Interest in this protein stems largely from the fact that it induces an autoimmune condition in animals—experimental allergic encephalomyelitis—which serves as a model for the clinical demyelinating disease multiple sclerosis. This suggests that the A-1 protein interacts with circulating Antibodies in intact myelin, pointing to its localization On the surface of the myelin membrane. Nevertheless, its physiological function remains unknown.

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FIG. 5-2. A. Schematic model of the erythrocyte membrane. 1 — glycoprotein of molecular weight 100,000; 2 — sialoglycoprotein (glycophorin); 3 — spectrin; 4 — glyceraldehyde-3-phosphate dehydrogenase; 5 — (Na⁺+K⁺)-dependent ATPase; 6 — acetylcholinesterase. The proportions depicted in the figure do not reflect the actual stoichiometry of these polypeptide chains in the membrane. The rod-like structures (Y) represent carbohydrates. (Juliano R., BBA, 300, 341–348, 1973.) B. Schematic representation of a glycophorin molecule. Diamonds denote O-linked glycoside residues, and hexagons denote N-linked glycoside residues [31].

Another key protein component of myelin is a proteolipid heavily enriched in hydrophobic amino acid residues, which also contains Fatty acids presumably attached via ester linkages [9]. Similar proteolipids are quite widespread [33a]. A protein with a molecular weight of 12,000, soluble in a chloroform-methanol mixture (2:1), has been extracted from The Endoplasmic reticulum of Muscle cells. Subunits of E. coli F-pili (Chap. 1, Sec. A.6) of approximately the same size are located (in a solubilized state) in the outer membrane of the Introduction/37.html">Bacterial Cell wall [33b].

Many membrane proteins perform enzymatic Functions. For instance, the Mitochondrial Electron Transport system is localized within the membranes (Chap. 10), and several highly lipid-soluble enzymes have already been isolated. A case in point is phosphatidylserine decarboxylase, which catalyzes The conversion of phosphatidylserine to phosphatidylethanolamine (Chap. 12, Sec. E.2) during The Biosynthesis of the latter. Glycosyltransferases required for the biosynthesis of bacterial outer membrane lipopolysaccharides appear to be similarly embedded in The Plasma Membrane. These enzymes are inactive in the absence of Lipids, but their activity is restored upon The addition of phosphatidylethanolamine [34]. Of exceptional interest is a protein with a molecular weight of 26,000 found in the purple membranes of Halobacterium halobium (Chap. 13, Sec. E.6). This protein, whose structure has been elucidated by Electron Microscopy at a resolution of 0.7 nm, apparently functions as a light-driven proton pump [35]. Each protein molecule consists of seven closely packed α-helical segments oriented almost perpendicularly to the membrane plane. Together, these molecules form highly ordered arrays, with the intervening spaces filled with phospholipids.

This section provides a Brief Overview of current knowledge regarding the composition and architecture of cell membrane proteins. Without a doubt, future studies of membrane proteins will yield exciting new insights.