Photoaffinity labeling of atrial natriuretic factor receptors of rat kidney cortex plasma membranes

Synthetic rat atrial natriuretic factor (ANF) was derivatized with the N-hydroxysuccinimide ester of [125I]iodoazidosalicylic acid to yield a radioactive photoaffinity probe. Incubation of purified plasma membranes from rat kidney cortex with this photoaffinity probe resulted in the specific labeling of a 140-kDa glycoprotein. The photoaffinity labeling of this protein was inhibited by ANF but not by reduced and alkylated ANF nor by other unrelated peptides. A 140-kDa band was also specifically labeled in liver plasma membranes but not in adipocyte plasma membranes. These observations suggest strongly that the 140-kDa glycoprotein is the ANF receptor.     

Purification and characterization of a major glycoprotein in rat hepatoma plasma membranes. One of the membrane proteins released by phosphatidylinositol-specific phospholipase C.

A major glycoprotein of rat hepatoma plasma membranes was selectively released as a soluble form by incubating the membrane with phosphatidylinositol-specific phospholipase C. The soluble form corresponding to the glycoprotein was also prepared by butan-1-ol extraction of microsomal membranes at pH 5.5, whereas extraction at pH 8.5 yielded an electrophoretically different form with a hydrophobic nature. The soluble glycoprotein extracted at pH 5.5 was purified by sequential chromatography on concanavalin A-Sepharose, Sephacryl S-300 and anti-(alkaline phosphatase) IgG-Sepharose, the last step being used to remove a contaminating alkaline phosphatase. The glycoprotein thus purified was a single protein with Mr 130,000 in SDS/polyacrylamide-gel electrophoresis, although it behaved as a dimer in gel filtration on Sephacryl S-300. The glycoprotein was analysed for amino acid and carbohydrate composition. The composition of the carbohydrate moiety, which amounted to 64% by weight, suggested that the glycoprotein contained much larger numbers of N-linked oligosaccharide chains than those with O-linkage. It was confirmed that the purified glycoprotein was immunologically identical not only with that released by the phospholipase C but also with the hydrophobic form extracted with butan-1-ol at pH 8.5. The results indicate that the glycoprotein of rat hepatoma plasma membranes, which has an unusually high content of carbohydrate, is another membrane protein released by phosphatidylinositol-specific phospholipase C, as documented for alkaline phosphatase, acetylcholinesterase and Thy-1 antigen.     

Isolation of the major glycoprotein from plasma membranes of an ascites hepatoma AH 66.

Plasma membranes were isolated from AH 66 cells, some of which had been labeled with [14C]glucosamine, by the following procedure: homogenization of cells which had been hardened by treatment with Zn ions, fractionation of the homogenate by sucrose density gradient centrifugation and purification of the membranes by partition in an aqueous two-phase polymer system. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) of the plasma membranes and subsequent staining of the gel for protein and carbohydrate, and determination of radioactivity on the gel eluates indicated the presence of at least 10 bands of glycoproteins. The major band contained 27% of the total radioactivity incorporated into the plasma membranes and was most heavily stained with the periodate-Schiff reagent. To isolate the major glycoprotein, the membranes were solubilized with 0.6 M lithium diiodosalicylate containing 0.5% Triton X-100, then the solution was treated with phenol. The major glycoprotein, obtained in the aqueous phase, was further purified mainly by repeated chromatographies on Sepharose 6B. The purified preparation was practically homogeneous on SDS-polyacrylamide gel electrophoresis, as judged by radioactivity determination and by carbohydrate staining, but contained small amounts of carbohydrate-free proteins. The major glycoprotein had an apparent molecular weight of 160,000, as determined by SDS-polyacrylamide gel electrophoresis. The final preparation contained about 44% carbohydrate on a weight basis, and the carbohydrate moiety was composed of glucosamine, galactosamine, galactose, mannose, fucose, and sialic acid. This composition indicates that the major glycoprotein contains both N- and O-glycosidically linked oligosaccharide moieties.     

Isolation and partial characterization of the major glycoproteins of horse and swine erythrocyte membranes

The major glycoproteins of horse and swine erythrocyte membranes were isolated and examined chemically and immunologically. The major glycoprotein of horse erythrocyte membranes had a molecular weight of 33 000 and consisted of 46.2% protein and 53.8% carbohydrate, of which 9.4% was hexose, 10.1% hexosamine and 33.7% sialic acid. This glycoprotein was associated with activity for the infectious mononucleosis heterophile antigen.There were two different major glycoproteins in swine erythrocyte membranes. One major glycoprotein had a molecular weight of 46 200 and consisted of 34.2% protein and 65.8% carbohydrate, of which 18% was hexose, 19% hexosamine and 27.2% sialic acid. This glycoprotein had phytohemagglutinin (Phaseolus vulgaris) binding activity. The other glycoprotein had a molecular weight of 29 000 and consisted of 50.4% protein and 49.6% carbohydrate, of which 6.4% was hexose, 7.0% hexosamine and 36.3% sialic acid. This glycoprotein had weak or absent phytohemagglutinin binding activity.     

Development of monoclonal antibodies against different protein and carbohydrate epitopes of dipeptidyl peptidase IV from rat liver plasma membranes.

Dipeptidyl peptidase IV (DPP IV) is a serine exopeptidase expressed at high levels in rat kidney, liver and lung. We established eight monoclonal antibodies against partially purified DPP IV from rat liver plasma membranes. By means of a competitive dot blot assay with purified DPP IV, these antibodies were shown to recognize four different epitopes of the glycoprotein, designated A - D. The epitopes are located on the extracellular domain of DPP IV, as shown by papain digestion of liver plasma membranes. Treatment of DPP IV with neuraminidase and glycopeptide N-glycosidase F, as well as incubation of hepatocytes with the alpha-mannosidase I inhibitor deoxymannojirimycin, revealed that epitope A may be formed by a mannose-rich sugar chain and epitope D might represent a complex carbohydrate structure in the mature glycoprotein, while the epitopes B and C are formed by the protein moiety. Concanavalin A reduced the binding of monoclonal antibody to epitope A by 78%. Binding to epitope D was blocked by 73% with wheat germ lectin, and by more than 99% with sialic acid; epitopes B and C were unaffected by any of the lectins or sugars tested. The immunological cross-reactivity with DPP IV from Morris hepatoma 7777 was demonstrated with monoclonal antibodies against epitopes A-C. Epitope D was not recognized on hepatoma DPP IV. However, in addition to DPP IV, four hepatoma plasma membrane glycoproteins were precipitated by the monoclonal antibody against the epitope D, indicating that this epitope is not uniquely restricted to DPP IV.     

Chemical characterization of the proteins and glycoproteins of mouse liver plasma membranes solubilized by sequential extraction with aqueous and organic solvents

1. A procedure for the stepwise fractionation of the proteins of mouse liver plasma membranes is described. 2. Of the membrane protein 20-25% was soluble in 50mm-sodium carbonate-bicarbonate buffer (pH9.7). This fraction contained a large number of proteins but only 1 major glycoprotein. It was low in sialic acid, amino sugars and phospholipid. 3. Extraction of the alkali-insoluble residue with aq. 33% pyridine solubilized an additional 30-35% of the membrane protein. The pyridine-soluble membrane components were enriched in sialic acid and glucosamine and it was shown that this procedure resulted in the selective extraction of glycoproteins. 4. Gel filtration in sodium dodecyl sulphate resolved the pyridine-soluble proteins into five fractions of decreasing molecular weight and an inverse relationship between molecular weight and sialic acid content was indicated.     

Isolation of the major glycoprotein from fat cell plasma membranes

The major glycoprotein of rabbit fat cell plasma membranes has been solubilized by Brij 99 extraction and purified to homogeneity by preparative polyacrylamide gel electrophoresis and concanavalin A-Sepharose affinity chromatography. The isolation procedure yielded a glycoprotein with an apparent molecular weight of 79,000 which appeared as a single component by Coomassie blue and periodic acid-Schiff staining as well as by distribution of radioactivity after ¹²⁵I labeling. The lectin chromatography was effective in removing polypeptides and Schiff-nonreactive glycoproteins which migrated in close proximity to the major glycoprotein during electrophoresis but were not retained on the concanavalin A column. Determination of the amino acid and sugar composition of the purified glycoprotein indicated that it contained 18% carbohydrate by weight which occurred in the form of 30 mannose, 14 galactose, 23 glucosamine, 3 galactosamine, 6 N-acetylneuraminic acid, and 1 fucose residues per molecule. Approximately one-fifth of the total protein-bound saccharide of the adipocyte plasma membrane was accounted for by this glycoprotein and its composition suggested that it was the source of some of the previously identified (Y. Kawai, and R. G. Spiro, 1977, J. Biol. Chem.252, 6236–6244) asparagine- and serine (threonine)-linked carbohydrate units of the fat cell surface.     

Biological function of the carbohydrate component of human sex hormone-binding globulin

The role of the carbohydrate component of sex steroid-binding globulin (SBP) from human blood in the glycoprotein interaction with the recognition system for SBP-estrogen complexes in human decidual endometrium plasma membrane was studied. It was shown that the removal of N-acetylneuraminic acid residues from the oligosaccharide chains of SBP did not affect the steroid-binding or immunochemical properties of the glycoprotein. At the same time, the above modification of the glycoprotein resulted in a loss by SBP of its ability to specifically interact with the membrane recognition system. It is concluded that the oligosaccharide chains of SBP are involved in the formation of determinants needed for recognition of the SBP-estrogen complexes by endometrium cell plasma membranes.     

Mannosyl transferases inSaccharomyces cerevisiae: Evidence for the occurrence of ectomannosyltransferase activity

The subcellular distribution of mannosyltransferases inSaccharomyces cerevisiae was studied following the separation of the plasma membrane from other intracellular membranous systems. Most of the activity was linked to internal membranes, and the rest was located at the level of the plasma membrane. Yeast plasma membranes coated on their external face with concanavalin A when incubated with GDP-[U-¹⁴C]mannose incorporated 20% less [U-¹⁴C]mannose in glycoproteins and 110% more in glycolipids than plasma membranes alone. This suggested that part of the total mannosyltransferase activity of the plasma membrane is located on its outer surface. A significant incorporation of radioactive mannose into trichloroacetic-acid-precipitable material was detected in incubations of protoplasts with GDP-[U-¹⁴C]mannose when incorporation of free mannose did not occur. Characterization of a product synthesized by the ectotransferase(s) was achieved after treatment of the radioactive plasma membranes by Triton X-100, which preserved the concanavalin A-mannoprotein complexes and removed a large amount of other plasma membrane components. A radioactive glycoprotein band with an apparent molecular weight of 94, 000 was identified as a product of the ectomannosyltransferase(s).     

Behavior of vesicular stomatitis virus glycoprotein in mouse LM cells with modified membrane-phospholipids

LM cells in which the membrane phospholipids had been modified with choline analogues were infected with vesicular stomatitis virus. The choline analogues tested were choline, N,N'-dimethylethanolamine, N-monomethylethanolamine and ethanolamine. These modifications per se did not affect the syntheses of individual viral proteins. The viral glycoprotein was detected in the plasma membranes of all the modified cells by pronase digestion in pulse-chase experiments, but the amount of glycoprotein susceptible to proteolysis varied, decreasing in these modified cells in the following order: N,N'-dimethylethanolamine- greater than choline- greater than N-monomethylethanolamine- greater than ethanolamine-treated cells. After a 4-h chase, glycoprotein was mainly distributed in the plasma membranes of cells modified with N,N'-dimethylethanolamine, whereas it was found in both the microsomes and plasma membranes of cells modified with other analogues. Fairly large amounts of glycoprotein were also found in the soluble fraction of ethanolamine-treated cells, but not in that of choline- or N,N'-dimethylethanolamine-treated cells. More precise experiments on the behaviour of glycoprotein with a short period of chase strongly suggested that migration of glycoprotein from the microsomes to the plasma membranes was fastest in cells modified with N,N'-dimethylethanolamine and slowest in cells modified with ethanolamine. Membrane lipid modifications also resulted in release of different numbers of progeny virions from the cells, release of virions from the cells decreasing in the following order: N,N'-dimethylethanolamine- greater than choline- greather N-monomethylethanolamine- greater than ethanolamine-treated cells. These results indicate that modification of membrane phospholipids influences not only the insertion of glycoprotein into the microsomes and its migration to the plasma membranes, but also the production of progeny virions.     

Isolation and characterization of plasma-membrane glycoproteins from pig epidermis

1. Non-desmosomal plasma membranes enriched in plasma-membrane marker enzymes and in metabolically labelled glycoproteins were isolated on a large scale from up to 500g of pig ear skin slices. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and periodic acid/Schiff staining revealed the presence of four major glycosylated components in the apparent molecular-weight range 150000–80000. 2. A large proportion of the marker enzymes, the d-[³H]glucosamine-labelled glycoproteins and the periodic acid/Schiff-stained glycoproteins were solubilized by 1% (w/v) sodium deoxycholate. However, several non-glycosylated proteins, in particular those with mol.wts. 81000, 41000 and 38000 (possibly cytoskeletal components), were relatively resistant to solubilization. 3. The deoxycholate-solubilized membranes were fractionated by lectin affinity chromatography using both concanavalin A–Sepharose 4B and lentil lectin–Sepharose 4B. From 75 to 85% of the applied glycoprotein was recovered from the columns. From 30 to 40% of the recovered glycoprotein was specifically bound by the lectins and was eluted with 2% (w/v) α-methyl d-mannoside. The enrichment of labelled glycoproteins in the material bound by the lectins (2.5-fold) was similar with both lectins, although the yield was somewhat greater when lentil lectin was used. The glycoprotein-enriched fraction was also enriched in all the plasma-membrane marker enzymes, indicating their probable glycoprotein nature. 4. The glycoprotein-enriched fraction contained the four major periodic acid/Schiff-stained bands that were detected in the original plasma membrane. They had apparent mol.wts. 147000, 130500, 108000 and 91400. The higher-molecular-weight components contained relatively more d-[³H]glucosamine, indicating differences in the sugar composition or in the metabolic turnover of the individual glycoproteins in culture. The material bound by the lectins also contained a number of lower-molecular-weight Coomassie Brilliant Blue-stained components. These were weakly stained by periodic acid/Schiff reagent and were lightly labelled with d-[³H]glucosamine, indicating that they contained less carbohydrate than the four major glycoprotein bands. 5. Chloroform/methanol-extracted plasma membranes and isolated glycoproteins had a similar carbohydrate composition, containing sialic acid, hexosamine, fucose, xylose, mannose, galactose and glucose. Glucose was not enriched in the isolated glycoproteins, suggesting that it may be a contaminant. Xylose, however, was enriched in the isolated glycoproteins. It remains to be established whether this sugar, which is not usually found in plasma-membrane glycoproteins, is a genuine constituent of plasma-membrane glycoproteins in the epidermis.     

Delineation and synthesis of the membrane receptor-binding domain of sex hormone-binding globulin

Sex hormone-binding globulin (SHBG) is a plasma glycoprotein which binds certain steroids. It, in turn, binds to a specific receptor on cell membranes. This work was undertaken to identify, isolate, sequence, and synthesize the region of SHBG that interacts with its membrane receptor. To accomplish this, highly purified human SHBG was digested with trypsin. The SHBG-derived tryptic peptides were separated by high performance liquid chromatography. They were evaluated for their ability to compete with 125I-SHBG for binding to the SHBG receptor solubilized from human prostatic membranes. Only a single peptide, corresponding to residues 48-57 of the known sequence of human SHBG, inhibited receptor binding. A synthetic decapeptide with this amino acid sequence also competitively inhibited SHBG binding.     

Vesicular stomatitis virus glycoprotein does not determine the site of virus release in polarized epithelial cells

In polarized epithelial cells, the vesicular stomatitis virus glycoprotein is segregated to the basolateral plasma membrane, where budding of the virus takes place. We have generated recombinant viruses expressing mutant glycoproteins without the basolateral-membrane-targeting signal in the cytoplasmic domain. Though about 50% of the mutant glycoproteins were found at the apical plasma membranes of infected MDCK cells, the virus was still predominantly released at the basolateral membranes, indicating that factors other than the glycoprotein determine the site of virus budding.     

Purification and properties of the major sialoglycoprotein of the milk fat globule membrane

The major periodate-Schiff positive component (glycoprotein-2) of bovine milk fat globule membranes (MFGM) has been purified by extraction of washed cream with chloroform/methanol followed by chromatography on Sephadex G-200 in sodium dodecyl sulfate. The glycoprotein is > 95% pure by polyacrylamide electrophoresis in dodecyl sulfate and shows the same prominent component at gel percentages of from 5 to 12.5. The molecular weight obtained by extrapolation of the apparent molecular weights on these gels to higher gel percentages was 70,000. An apparent molecular weight of 105,000 was obtained by gel filtration in 1% dodecyl sulfate on Sepharose 4B. The glycoprotein contains 50% carbohydrate by weight, with sialic acid (30.5%), N-acetylglucosamine (22.3%), galactose (15.9%), N-acetylgalactosamine (14.0%), mannose (11.1%), and fucose (5.8%) being the major monosaccharides. Leucine, glutamic acid, and glycine are the major amino acids. Affinity chromatography of deoxycholate-solubilized MFGM indicates that glycoprotein-2 is not the major concanavalin A receptor of these membranes.     

Lysosomal membrane dynamics: structure and interorganellar movement of a major lysosomal membrane glycoprotein

The biochemistry and intracellular transit of an integral membrane glycoprotein of chicken fibroblast lysosomes were studied with monoclonal antibody techniques. The glycoprotein had an apparent molecular weight of 95,000-105,000. Structural analysis involving metabolic labeling with [35S]methionine and cleavage with glycosidases revealed the presence of numerous oligosaccharide chains N-linked to a core polypeptide of apparent molecular weight 48,000. A primary localization of the glycoprotein to lysosomes was demonstrated by the coincidence of antibody binding sites with regions of acridine orange uptake, electron immunocytochemical labeling on the inner surface of lysosome-like vacuolar membranes, and preferential association of the glycoprotein with lysosome-enriched subcellular fractions from Percoll gradients. In addition, small quantities of the glycoprotein were detected on endocytic vesicle and plasma membranes. To study the intracellular pathway of the glycoprotein, we used a monoclonal antibody whose binding to the glycoprotein at the cell surface had no effect on the number or subcellular distribution of antigen molecules. Incubation of chicken fibroblasts with monoclonal antibody at 37 degrees C led to the rapid uptake and subsequent delivery of antibody to lysosomes, where antibody was degraded. This process continued undiminished for many hours on cells continuously exposed to the antibody and was not blocked by the addition of cycloheximide. The rate at which antigen sites were replenished in the plasma membrane of cells prelabeled with antibody (t1/2 = 2 min) was essentially equivalent to the rate of internalization of antibody bound to cell surfaces. These results suggest that there is a continuous and rapid exchange of this glycoprotein between plasma membrane and the membranes of endosomes and/or lysosomes.     

Purification and characterization of a probable light receptor with kinase activity from beet root plasma membranes

A 120-kDa glycoprotein was found in beet root (Beta vulgaris L.) plasma membranes. This protein could be phosphorylated in a Ca2+-independent manner. Its carbohydrate moiety was composed of both O-linked galactose-beta(1-3)-N-acetylgalactosamine disaccharides (which bind peanut agglutinin) and N-linked concanavalin A (ConA)-binding oligosaccharides. The phosphorylation of this protein was rapid, half-saturated with 6 microM ATP and higher at alkaline pH values. This protein was phosphorylated more efficiently with Mn-ATP as substrate than with Mg-ATP. This phosphorylation increased when plasma membranes were illuminated with low-fluence blue light, a fact suggesting that the 120-kDa glycoprotein could be similar to phototropin: a blue-light photoreceptor involved in phototropism. This protein was purified using a ConA-Sepharose column. The phosphorylation of the purified protein could be observed, but it was much lower than that of the 120-kDa protein in plasma membranes. In addition, it was not enhanced by light. Some possible explanations for this photosensitivity loss are discussed.     

Decreased binding of insulin to liver plasma membrane receptors in hereditary diabetic mice.

The interaction of insulin with its receptors was studied in liver plasma membranes of the young non-obese hereditary diabetic mouse (KK strain). Under identical conditions of preparation and incubation, the membranes of the KK mouse bind only 55-70% as much insulin per mg of protein as those of the control mouse (Swiss albino). Scatchard analysis suggests that this decrease in binding is due to a decrease in the number of receptor sites in the membrane of the diabetic mouse. However, the membranes of diabetic and control mice do not exhibit significant differences in hexosamine and sialic acid contents, enzyme activities, and protein and glycoprotein analysis. The decrease in insulin receptors in the KK mouse seems to correlate with the insulin resistance which they exhibit.     

Characterization of the cryptogein binding sites on plant plasma membranes

Cryptogein is a 98-amino acid proteinaceous elicitor of tobacco defense reactions. Specific binding of cryptogein to high affinity binding sites on tobacco plasma membranes has been previously reported (K(d) = 2 nM; number of binding sites: 220 fmol/mg of protein). In this study, biochemical characterization of cryptogein binding sites reveals that they correspond to a plasma membrane glycoprotein(s) with an N-linked carbohydrate moiety, which is involved in cryptogein binding. Radiation inactivation experiments performed on tobacco plasma membrane preparations indicated that cryptogein bound specifically to a plasma membrane component with an apparent functional molecular mass of 193 kDa. Moreover, using the homobifunctional cross-linking reagent disuccinimidyl suberate and tobacco plasma membranes incubated with (125)I-cryptogein, we identified, after SDS-polyacrylamide gel electrophoresis and autoradiography, two (125)I-cryptogein linked N-glycoproteins of about 162 and 50 kDa. Similar results were obtained using Arabidopsis thaliana and Acer pseudoplatanus plasma membrane preparations, whereas cryptogein did not induce any effects on the corresponding cell suspensions. These results suggest that either cryptogein binds to nonfunctional binding sites, homologues to those present in tobacco plasma membranes, or that a protein involved in signal transduction after cryptogein recognition is absent or inactive in both A. pseudoplatanus and A. thaliana.     

Glycoprotein synthesis in the adult rat pancreas. IV. Subcellular distribution of membrane glycoproteins

Zymogen granule membranes from the rat exocrine pancreas displays distinctive, simple protein and glycoprotein compositions when compared to other intracellular membranes. The carbohydrate content of zymogen granule membrane protein was 5-10-fold greater than that of membrane fractions isolated from smooth and rough microsomes, mitochondria and a preparation containing plasma membranes, and 50-100-fold greater than the zymogen granule content and the postmicrosomal supernate. The granule membrane glycoprotein contained primarily sialic acid, fucose, mannose, galactose and N-acetylglucosamine. The levels of galactose, fucose and sialic acid increased in membranes in the following order: rough microsomes less than smooth microsomes less than zymogen granules. Membrane polypeptides were analyzed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The profile of zymogen granule membrane polypeptides was characterized by GP-2, a species with an apparent molecular weight of 74 000. Radioactivity profiles of membranes labeled with [3H]glucosamine or [3H]leucine, as well as periodic acid-Schiff stain profiles, indicated that GP-2 accounted for approx. 40% of the firmly bound granule membrane protein. Low levels of a species similar to GP-2 were detected in membranes of smooth microsomes and the preparation enriched in plasma membranes but not in other subcellular fractions. These results suggest that GP-2 is a biochemical marker for zymogen granules. Membrane glycoproteins of intact zymogen granules were resistant to neuraminidase treatment, while those in isolated granule membranes were readily degraded by neuraminidase. GP-2 of intact granules was not labeled by exposure to galactose oxidase followed by reduction with NaB3H4. In contrast, GP-2 in purified granule membranes was readily labeled by this procedure. Therefore GP-2 appears to be located on the zymogen granule interior.     

Isolation and biochemical characterization of the alpha- and beta-subunits of glycoprotein IIb of human platelet plasma membrane.

The alpha- and beta-subunits of glycoprotein IIb (GPIIb) of human platelet plasma membrane were isolated in fully reduced, partially reduced and alkylated, and fully alkylated forms, by size-exclusion chromatography after reduction of pure GPIIb. The sugar moiety of GPIIb alpha accounts for 16.4% of its total weight, whereas that of GPIIb beta accounts for only 10.2%. The molar percentages (per 100 mol of total amino acids) of neuraminic acid and galactose in the alpha-subunit more than double those in the beta-subunit, whereas galactosamine is present only in GPIIb alpha. From the amino acid and sugar compositions the acidic nature of both subunits was confirmed. The Mr values obtained, 114,000 for GPIIb alpha and 22,200 for GPIIb beta, are in very good agreement with those obtained by physical methods. We found by stepwise reduction of pure GPIIb with dithioerythritol that GPIIb alpha and GPIIb beta are joined by a single interchain disulphide bridge, while the remaining half-cystine residues participate in intrachain bonds, six in GPIIb alpha and one in GPIIb beta, the intersubunit disulphide bond being that reduced first. Neither of the two subunits is liberated from isolated plasma membranes when this GPIIb interchain bond is reduced in isolated membranes.