Intrinsic Membrane Glycoprotein Research Articles

Structure of human platelet membrane glycoproteins IIb and IIIa as determined by electron microscopy.

The glycoprotein (GP) IIb-IIIa complex was isolated from human platelet membranes and examined for glycoprotein stoichiometry and morphology. To determine the ratio of glycoproteins in the complex, the isolated glycoproteins were solubilized with sodium dodecyl sulfate and separated by high-performance liquid chromatography. Quantitative amino acid analysis of individual glycoproteins showed that the ratio of GP IIb to GP IIIa in the Ca2+-dependent complex was 0.93:1. Morphology was determined by electron microscopy of rotary-shadowed and negatively stained specimens. Individual complexes consisted of two domains: an oblong head of approximately 8 X 10 nm with two rodlike tails extending approximately 14-17 nm from one side of the head. Treatment of the isolated complex with EDTA resulted in the appearance of a mixture of oblong and filamentous structures, which could be separated by a sucrose gradient sedimentation in Triton X-100. As seen by rotary and unidirectional shadowing, GP IIb was a compact structure, approximately 8 X 10 nm in size. Isolated GP IIIa was more heterogeneous but was most often observed in an elongated form, varying in length from 20 to 30 nm and in width from 2 to 3 nm. By comparing these structures to that of the heterodimer complex, it was determined that the oblong domain was GP IIb and the rodlike tails were GP IIIa. Each milligram of isolated GP IIb-IIIa complex bound 0.30 mg of [3H]Triton X-100, indicating that the glycoprotein complex contained limited hydrophobic domains. Upon removal of detergent, GP IIb-IIIa complexes formed aggregates that sedimented in sucrose gradients as a diffuse peak ranging from 14 to 32 s. Examination of these aggregates by electron microscopy showed that they were composed of clusters or "rosettes" of 2 to 20 or more of the GP IIb-IIIa complexes. The orientation of these rosettes was such that the tails were joined in the center, with the head portions directed away from the interacting tails. It thus appears that the primary hydrophobic domains of the GP IIb-IIIa complex exist at the tips of the GP IIIa tails. Because the GP IIb-IIIa complex is an intrinsic membrane glycoprotein, these findings indicate a potential membrane attachment site for the GP IIb-IIIa complexes.

Reactivity with lectins of the saccharide components of rhodopsin in reconstituted membranes. Orientation of the carbohydrates

Rhodopsin-containing liposomes may provide a model for investigating the interaction of intrinsic membrane glycoproteins in biological systems. As part of the characterization of this preparation, the surface orientation of the carbohydrates of rhodopsin, assembled from purified bovine rhodopsin and egg phosphatidylcholine was examined, and is the topic of this report. The major tool used in these studies was the interaction with the carbohydrate-specific reagents, plant lectins. Two techniques were used: (1) lectin-mediated aggregation of the liposomes, as measured by light scattering; (2) the binding of 125I-labeled succinylated concanavalin A, and Scatchard analysis as a measure of affinity. The preparation most extensively examined had a mole ratio of rhodopsin:phospholipid of 1:100. Among a variety of lectins which were examined, only concanavalin A, succinylated concanavalin A, and wheat germ agglutinin were able to mediate the aggregation of rhodopsin-containing liposomes, as expected. The aggregation with concanavalin A was prevented by the presence of sugars having the α- d-glucopyranosyl configuration, and that brought about with wheat germ agglutinin, by N-acetylglucosamine (GlcNAc). In addition, the aggregation with concanavalin A was reversed with methyl α- d-mannoside, and with wheat germ agglutinin, by GlcNAc, suggesting that membrane fusion did not take place. On a molar basis, wheat germ agglutinin brought about a greatly reduced extent of aggregation as compared to concanavalin A, suggesting the relative inaccessibility of GlcNAc residues in the liposomes as compared to mannose. The initial rate of the aggregation, however, were similar with either lectin. The first-order rate constants were unaffected by wide variation in the concentrations of concanavalin A and wheat germ agglutinin, and by variation in the mole ratios of rhodopsin in the liposomes from 0.2 to 19 moles per 100 moles of egg lecithin. Rhodopsin-liposomes were also prepared from a total lipid extract of rod outer segments instead of egg lecithin. Similar kinetic properties were exhibited by this preparation as were obtained with the liposome prepared with the purified phospholipid. Scatchard analysis of the binding of 125I-labeled succinylated concanavalin A by rhodopsin liposomes indicated the presence of a single class of binding site as the preferred fit, with an apparent K d of 2.8·10 −7 M. The binding was destroyed or extensively interfered with by trypsinization and by periodate treatment.

Insulin-Dependent Tyrosine Protein Kinase: Cellular Distribution and Copurification with the Insulin Receptor

Publisher Summary The first step in insulin action involves the binding of insulin to a class of specific, high-affinity receptors on the cell surface. The receptor is an intrinsic membrane glycoprotein that contains two kinds of subunits—alpha and beta. An additional or alternative model for the mechanism of action of insulin was suggested by recent discoveries that the insulin receptor in intact cells becomes phosphorylated on tyrosine and serine residues in response to insulin and that insulin-dependent tyrosine protein kinase activity can be demonstrated in vitro in preparations containing the insulin receptor. This chapter focuses on three properties of the insulin-dependent tyrosine protein kinase: (1) evidence that the insulin receptor isolated from a variety of tissues is immunologically conserved and associated with a constant ratio of insulin dependent tyrosine protein kinase activity to high-affinity insulin-binding activity, (2) copurification to homogeneity of the human placental insulin-dependent tyrosine protein kinase and insulin receptor, and (3) evidence that phosphorylation modifies the properties of the protein kinase intrinsic to the insulin receptor. The insulin-dependent tyrosine protein kinase can catalyze the phosphorylation of tyrosine residues in certain proteins and peptides.

Monoclonal antibodies to rabbit sperm autoantigens. I. Inhibition of in vitro fertilization and localization on the egg

Biochemical and antigenic similarities exist among members of what can be considered a family of low molecular weight rabbit sperm autoantigens. These autoantigens are intrinsic plasma membrane glycoproteins specific to spermatogenic cells and spermatozoa. The amino acid and carbohydrate compositions of rabbit sperm autoantigen-1 (RSA-1) and RSA-2 were compared and monoclonal antibodies (mAb) were raised in mice against rabbit sperm autoantigens. The epitopes recognized by the antibodies were present on RSA-1, 2 and 3. A monoclonal anti-RSA-1, 2 and 3 (designated A.F. 1) was used to localize the antigen on spermatozoa and testis cells and investigate the epitope's tissue specificity. This mAb inhibited in vitro fertilization but did not block the sperm from dispersing the cumulus cells surrounding the egg. The mAb also demonstrated the presence of RSA-1, 2 and 3 on the plasma membrane of the egg after fertilization. It is concluded that the RSA family plays a central role in zona penetration.

Subcellular Localization of the Hepatic Asialoglycoprotein Receptor by Immunoelectron Microscopy

The hepatic receptor for asialoglycoproteins provides an excellent system with which to study the subcellular localization and function of an intrinsic membrane glycoprotein. This receptor, specific for galactoseterminal glycoproteins, mediates the rapid and specific binding and subsequent internalization of glycoprotein ligands. The receptor is abundant; there are approximately 500,000 high affinity ligand binding sites per rat hepatocyte cell surface. The receptor has been isolated from rat liver by affinity chromatography. Using a monoclonal antibody, we showed that the surface receptor consists of two non-identical subunits which contain the same or closely related polypeptide chains. Morphologic studies, primarily by A. Hubbard and collaborators, have evaluated the fate of the asialoglycoprotein ligand once bound by the hepatocyte. They demonstrated, both in the rat and in perfused rat liver, a rapid association of labeled ligand with the plasma membrane of the parenchymal cell. This was followed by accumulation of ligand within the lysosomes and Golgi region. We have taken an immunocytochemical electron microscopic approach to examine the distribution and localization of the asialoglycoprotein receptor both in normal rat liver, and in liver cells exposed to asialoglycoprotein ligand.

Photoaffinity labeling of the adenosine transport system in adipocyte plasma membranes

A membrane component involved in the transport of adenosine in adipocytes has been identified utilizing the techniques of photoaffinity labeling with the adenosine derivative, 8-azidoadenosine. In the absence of light, adenosine and 8-azidoadenosine exhibited similar transport characteristics. In addition, adenosine was shown to be a competitive inhibitor of 8-azidoadenosine uptake, and the photoprobe, a competitive inhibitor of adenosine uptake. Analysis of the nucleotide metabolites indicated that the photoprobe was metabolized in a similar fashion to that observed for adenosine. Several nucleoside transport inhibitors were also equally effective in inhibiting the uptake of both nucleosides. These results suggest that 8-azidoadenosine is transported by the same membrane system as adenosine. Photolysis of 8-azido[2- 3H]adenosine in the presence of adipocytes resulted in the covalent incorporation of the photoprobe into the plasma membrane fraction. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that essentially all of the radioactivity was incorporated into a glycoprotein with a molecular weight of 56,000. This labeling was inhibited by greater than 90% when the photolysis was carried out in the presence of excess adenosine or the transport inhibitors, persantin or theophylline. Fractionation of the labeled plasma membranes by dialysis against water (pH 9.5) indicated that approximately 75% of the radioactivity was associated with a glycoprotein which resisted solubilization by this procedure. These results suggest that the major labeled species is a 56,000 M r intrinsic membrane glycoprotein which may function as a component of a transmembrane assembly involved in the transport of adenosine.

36] Isolation and purification of bovine rhodopsin

Publisher Summary This chapter describes the isolation and purification of bovine rhodopsin. Rhodopsin is the general name for the visual pigments of both the rod cells in the vertebrate retina and the invertebrate photoreceptor cells. Rhodopsin is an intrinsic membrane glycoprotein, confined to the membranous structures (photoreceptor membranes) of a special part of the rod cell, the rod outer segment (ROS), which is highly specialized for light reception. Rhodopsin is determined by measuring the decrease in absorbance at 500 nm upon illumination, using a molar absorbance of 40,500. Under some conditions, the photoproducts also absorb at 500 nm, but addition of hydroxylamine will convert all free and bound retinaldehyde into retinal oxime, which has virtually no absorption beyond 450 nm. In detergents like the quarternary ammonium derivatives (DTAB, TTAB, CTAB) or the amine oxides (C10DAO, C12DAO), addition of hydroxylamine is not required, because under these conditions retinal is completely released and hardly interferes at 500 nm. Hydroxylamine should be handled with care, because it is highly mutagenic. The assay can be performed on suspensions of photoreceptor membranes, but scattering artifacts may interfere. Most accurate results are obtained after prior solubilization of the membrane in an appropriate detergent, followed by centrifugation or filtration through a membrane filter if some turbidity remains.

Structural analysis of human platelet membrane glycoprotein I complex.

The glycoprotein I complex, consisting of two polypeptides of Mr 210,000 and 150,000, was isolated from human platelet membranes by wheat germ lectin affinity chromatography. Glycocalicin, a soluble loosely bound membrane glycoprotein of Mr 150,000 related to the glycoprotein I system, was also purified. The isolated polypeptides were radioiodinated in sodium dodecyl sulfate/polyacrylamide gels and digested with trypsin, and the labeled peptide digest was analyzed by two-dimensional high-voltage electrophoresis and thin-layer chromatography. The two polypeptides of Mr 210,000 and 150,000 in the glycoprotein I complex had essentially identical radioactive peptide maps. Glycocalicin had a completely different tryptic peptide map. These studies shed light on the molecular relationships of some of the components of the platelet membrane glycoprotein I system. The possibility is raised that the receptorlike function of the intrinsic platelet membrane glycoproteins may be related to the polymeric subunit associations of the constituent polypeptides.

Characterization and physiological function of rat renal gamma-glutamyltranspeptidase.

Rat renal gamma-glutamyltranspeptidase is an intrinsic membrane glycoprotein. The larger of its two subunits is apparently folded into two distinguishable domains which are separated by a protease-sensitive sequence of amino acids. Membrane binding of gamma-glutamyltranspeptidase results from the hydrophobic interaction of the nonpolar domain of the amphipathic subunit with the lipid bilayer. Localization of at least a portion of the gamma-glutamyl binding site on the smaller subunit limits the active site of the enzyme to one side of the membrane. Within the kidney, the enzyme is primarily associated with the luminal surface of the brush border membrane of the proximal straight tubule. Comparison of the kinetic properties of gamma-glutamyltranspeptidase with the pH and the substrates available within the tubular fluid suggests that the physiologically significant reaction catalyzed by the transpeptidase is the hydrolysis of glutathione and its S-derivatives. The glutathionemia and glutathionuria observed in a patient who lacks detectable gamma-glutamyltranspeptidase activity and in mice following specific inhibition of transpeptidase, support the hypothesis that the enzyme plays a major role in glutathione catabolism. It now appears that the activities attributed to the gamma-glutamyl cycle do not participate in amino acid transport, but instead constitute three separate metabolic pathways; the intracellular synthesis of glutathione, the intracellular degradation of gamma-glutamyl peptides and the extracellular hydrolysis of glutathione. The finding that various cells release reduced and oxidized glutathione indicates that glutathione turnover may be a process of intracellular synthesis, excretion and extracellular degradation.

Further studies on the protein composition and surface structure of normal platelets and platelets from patients with Glanzmann's thrombasthenia and Bernard-Soulier syndrome

Iodination followed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE) of platelets from a patient with Bernard-Soulier syndrome showed a normal distribution of radioactive label, and no gross abnormalities in the protein pattern could be observed. The absence of the easily soluble membrane glycoprotein, glycocalicin or GPS, and a reduced amount of the intrinsic membrane glycoprotein, GP I, thus seem to represent more specific abnormalities of those platelets than previously recognized. By examination of normal, iodinated platelets by SDS PAGE as described by Weber and Osborn (1) (conventional system), the bulk of radioactive label appeared in the 100,000 MW region, and was probably associated with the congruent PAS band (GP III). By incorporation of urea and EDTA in the gels, the main peak of radioactivity was seen in the 120,000 MW area (GP II). Very little radioactivity was incorporated into these proteins when thrombasthenic platelets were iodinated, and SDS PAGE in the conventional as well as the urea system revealed a strongly reduced intensity of the PAS band in the GP II region. It is concluded that the glycoprotein most heavily labeled by lactoperoxidase-catalyzed iodination migrated in the GP III region in the conventional system, and in the GP II region when examined in the urea system.