Direct evidence for the interaction of platelet surface membrane proteins GPIIb and III with cytoskeletal components: protein crosslinking studies

When intact platelets are incubated at 37 degrees C with Concanavalin A (ConA), the two major surface membrane proteins GPIIb and III become associated with the Triton-insoluble cytoskeleton. Preincubation of platelets with a variety of metabolic inhibitors, including cytochalasin B, 2-deoxy-D-glucose, and antimycin A or lidocaine, had no effect on the ability of ConA to produce this effect. These results suggested that the ConA-induced anchorage of GPIIb and III to the Triton-insoluble cytoskeleton is a passive process requiring clustering of GPIIb-III molecules but not requiring the metabolic energy of an intact cell. This was supported by experiments that showed that ConA binding to plasma membrane-rich fractions at 37 degrees C could induce association of GPIIb and III with a sedimentable actin-rich, Triton-insoluble membrane matrix. Similar results were obtained when membranes were first isolated from ConA-treated cells. Adding DNAse I, an actin depolymerizing agent, into the Triton extraction buffer inhibited the ConA-induced sedimentation of GPIIb-III and actin by 50% in the presence of Mg2+-ATP. Treatment of ConA-treated membranes with dimethyl-3,3'-dithiobispropiomidate, a bifunctional, reducible protein crosslinking agent, produced Triton-insoluble crosslinked species of discrete molecular weights. When these cross-linked species were analyzed by SDS-PAGE in the presence of beta-mercaptoethanol, they were found to be composed of a 180-200 K dalton protein, GPIIb, GPIII, and actin. Crosslinking of these components was equally effective after Triton treatment and indicated as well that the species crosslinked in the intact membrane was stable after Triton extraction. Addition of crosslinker to membranes not treated with ConA produced similar crosslinked species. Analysis of their composition on reduced gels revealed that the amounts of GPIIb and III were reduced greatly (less than 10% of the total input GPIIb and III) but that the 180-200 k dalton protein and actin content were similar to that seen with ConA-treated membranes. These results are consistent with the notion that ConA clusters mobile, unanchored molecules of GPIIb-III (approximately 90-95% of the total) around a small fraction of IIb-III that is associated with a submembranous cytoskeleton.     

Concanavalin A induces interactions between surface glycoproteins and the platelet cytoskeleton

We have measured the association of platelet surface membrane proteins with Triton X-100 (Triton)-insoluble residues in platelets surface labeled with 125I. In both concanavalin A (Con A)-stimulated and resting platelets, this fraction is composed largely of polypeptides with apparent molecular weights of 45,000, 200,000, and 250,000 which comigrate with authentic actin, myosin heavy chain, and actin binding protein, respectively, as judged by PAGE in SDS. Less than 10% of the two major 125I-labeled surface glycoproteins, GPiib and GPIII, were associated with the Triton residue in resting platelets. Within 45 s after Con A addition, 80-95% of these two glycoproteins became associated with the Triton residue and the amount of sedimentable actin doubled. No cosedimentation of GPIIb and III with the cytoskeletal protein-containing Triton residue was seen when Con A was added to a Triton extract of resting cells, indicating that the sedimentation of GPIIb and III seen in Con A-stimulated platelets was not due to precipitation of the glycoproteins by Con A after detergent lysis. Treatment of Triton extracts of Con A-stimulated platelets with DNase I (deoxyribonucleate 5'-oligonucleotidido-hydrolase [EC 3.1.4.5]) inhibited the sedimentation of actin and the two surface glycoproteins in a dose-dependent manner. This inhibition of cosedimentation was not due to an effect of DNase I on Con A-glycoprotein interactions since these two glycoproteins could be quantitatively recovered by Con A- Sepharose affinity absorption in the presence of DNase I. When the Con A bound to the Triton residue was localized ultrastructurally, it was associated with cell-sized structures containing filamentous material. In intact cells, there was simultaneous immunofluorescent coredistribution of surface-bound Con A and myosin under conditions which induced a redistribution of platelet myosin. These data suggest that Con A can, in the intact platelet, induce physical interactions between certain surface glycoproteins and the internal cytoskeleton.     

Activated phosphoinositide 3-kinase associates with membrane skeleton in thrombin-exposed platelets.

Human platelets undergo a rapid, major reorganization of the cytoskeletal matrix upon exposure to thrombin, and accumulate 3-phosphorylated phosphoinositides in a protein kinase C (PKC)-dependent manner. These phosphoinositides have been suggested to be involved in actin polymerization/depolymerization. We reasoned that, if newly generated 3-phosphorylated phosphoinositide modulates cytoskeletal reorganization, a prerequisite for such action would be generation near cytoskeletal proteins. We have found that, after platelet activation, phosphatidylinositol 3-kinase and phosphatidylinositol(4)P 3-kinase activities, antibody-detectable phosphoinositide 3-kinase, and PKC become markedly and specifically enriched in a Triton X-100-insoluble cytoskeletal fraction that contains GPIIb/IIIa (integrin) and pp60c-src. The cytoskeletal fraction then accounts for up to 70% of total phosphoinositide 3-kinase activity, a function of recruited activated enzyme. These proteins are not occluded or directly associated with newly polymerized actin, since blockage by cytochalasin D of actin polymerization, and consequent inhibition of accumulation of about 40% of incremental protein and actin in this fraction, has no effect on its content of phosphoinositide 3-kinase, GPIIb/IIIa, pp60c-src, or PKC. Depolymerization of actin with DNase I, or inhibition of ligand binding to GPIIb/IIIa by RGDS, however, in combination with cytochalasin D, further depletes actin and significantly decreases sedimentability of GPIIb/IIIa as well as phosphoinositide 3-kinase, pp60c-src, and PKC, without inhibiting total 3-kinase activity. Our results suggest that, as a function of platelet activation, enzymes that regulate the synthesis of 3-phosphorylated phosphoinositides rapidly associate with the membrane skeleton and that skeletally associated phosphoinositide 3-kinase is more active than the Triton-soluble form.     

Synaptic junctional glycoconjugates from chick brain

Forebrains from day-old chicks were homogenized and fractionated by differential sedimentation and density gradient centrifugation to yield subcellular fractions. The synaptosomal plasma membrane fraction was further treated with Triton X-100 to yield subsynaptic membrane fractions including synaptic junctions. Glycoproteins from these subsynaptic membrane fractions were identified after separation by SDS-polyacrylamide gel electrophoresis by incubating the gel slabs with radioiodinated concanavalin A. Two lectin-binding proteins were discerned in the synaptic junction fraction while none were observed in the Triton-soluble portion of the synaptic plasma membrane. The carbohydrate content of the glycoproteins from each subcellular fraction was quantitated after methanolysis and derivatization aso-methyl-trifluoroacetyl analogs by gas-liquid chromatography. The lowest concentration of glycoprotein sugars was found in the synaptic junction, mitochondrial, and soluble fractions while the greatest concentration was found in the myelin, light-synaptic plasma membrane, and the Triton-soluble portion of the synaptic plasma membrane. Of the subcellular fractions, the synaptic junction contained the highest porportion of mannose and lowest proportion of sialic acid. Moreover, this fraction's content of galactose andN-acetylglucosamine, relative to mannose was the lowest while its content of fucose was low. The oligosaccharide chains extending into the synaptic cleft therefore are predominantly of the neutral, mannose-rich type and are attached to a limited number of high-molecular-weight glycoproteins.     

A large-scale procedure for the isolation of integrin GPIIb/IIIa, the human platelet fibrinogen receptor

The heterodimer GPIIb/IIIa, formed by the Ca(2+)-dependent association of glycoproteins IIb (GPIIb) and IIIa (GPIIIa), is the major integrin at the platelet surface, where it serves as the receptor for fibrinogen and other adhesive proteins and plays a central role in platelet aggregation and in platelet adhesion to the subendothelium. Here we describe a procedure for the isolation of GPIIb/IIIa using as starting material either the whole particulate fraction, obtained by differential centrifugation after hypoosmotic lysis of glycerol-loaded platelets, or any of the fractions obtained by density gradient centrifugation of the whole particulate fraction. The procedure consists simply of differential extraction with Triton X-100 of the starting particulate fraction, anion-exchange chromatography of the 4% Triton X-100 supernatant, and size-exclusion chromatography of the GPIIb/IIIa-rich fraction retained in the ion-exchange column. The use of particulate fractions instead of whole platelets as the starting material for extraction together with differential extraction with Triton X-100 (two steps that are simple and inexpensive to perform) results in the early removal of many unwanted proteins, which otherwise would have to be removed at later stages at the expense of severely impairing the final yield of GPIIb/IIIa. Pure GPIIb/IIIa is obtained with a yield of about 48%, the highest so far reported, calculated with respect to the GPIIb and GPIIIa content in the starting particulate fraction. The final product can be stored in freeze-dried form without apparent changes in its physical and chemical properties.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of Hypothyroidism on Different Forms of Actin in Rat Cerebral Neuronal Cultures Studied by an Improved DNase I Inhibition Assay

An improved DNase I inhibition assay for the filamentous actin (F-actin) and monomeric actin (G-actin) in brain cells has been developed. Unlike other methods, the cell lysis conditions and postlysis treatments, established by us, inhibited the temporal inactivation of actin in the cell lysate and maintained a stable F-actin/G-actin ratio for at least 4-5 h after lysis. The new procedure allowed separate quantitation of the noncytoskeletal F-actin in the Triton-soluble fraction (12,000 g, 10 min supernatant) that did not readily sediment with the Triton-insoluble cytoskeletal F-actin (12,000 g, 10 min pellet). We have applied this modified assay system to study the effect of hypothyroidism on different forms of actin using primary cultures of neurons derived from cerebra of neonatal normal and hypothyroid rats. Our results showed a 20% increase in the Triton-insoluble cytoskeletal F-actin in cultures from hypothyroid brain relative to normal controls. In the Triton-soluble fraction, containing the G-actin and the noncytoskeletal F-actin, cultures from hypothyroid brain showed a 15% increase in G-actin, whereas the F-actin remained unaltered. The 10% increase in total actin observed in this fraction from hypothyroid brain could be totally accounted for by the enhancement of G-actin. The mean F-actin/G-actin ratio in this fraction was about 30% higher in the cultures from normal brain compared to that of the hypothyroid system, which indicates that hypothyroidism tends to decrease the proportion of noncytoskeletal F-actin relative to G-actin.     

The effects of proteases on proteins and glycoproteins of Dictyostelium discoideum plasma membranes

Dictyostelium discoideum cells were incubated with proteases, the plasma membranes subsequently isolated and changes in proteins and glycoproteins examined with dodecylsulfate gel electrophoresis. Low papain concentrations gave rise to a protein band which apparently derived from actin. Since actin was the only protein attacked, the results suggest some part of the actin is exposed on the outer surface of the cell. Higher papain concentrations released a substantial portion of actin from the plasma membrane and partially digested some of the glycoproteins. Since the new actin-derived band was not further digested, the glycoproteins may be required to stabilize the actin polymer rather than anchor those actin molecules which are directly associated with the plasma membrane. Pronase treatment released the two myosin heavy chains from the plasma membrane, in particular the higher molecular weight chain. Actin was not affected. Some glycoproteins were digested. Trypsin attacked many of the plasma membrane proteins, and the myosin heavy chains were completely removed. Actin was only moderately affected. However, the glycoproteins were entirely resistant to trypsin. Apparently the myosin heavy chains are attacked either due to their partial exposure on the cell surface or the exposure of proteins which anchor them in the membrane. These anchoring proteins cannot be glycoproteins or actin. Proteins and glycoproteins were largely digested when isolated plasma membranes were incubated with papain and pronase. The effects of trypsin on whole cells and isolated plasma membranes were similar.     

Actin in B16 melanoma cells of differing metastatic potential : Effects of trypsin and serum

Actin is present in cells in monomeric and polymeric (filamentous) forms. Filamentous actin is distributed in Triton-soluble (cytosolic) and Triton-insoluble (cytoskeletal core) fractions. We have used the DNase 1 inhibition assay and immunofluorescence to investigate the distribution of actin in monomeric and polymeric forms in cloned B16 murine melanoma cell lines of low and high metastatic capacity. The protease trypsin caused rounding up and detachment of both cell lines within 5 min. This was associated with almost complete depolymerization of cytosolic actin filaments but the Triton-insoluble cytoskeleton was not quantitatively affected by trypsin treatment. There were quantitative differences between the clones in their response to incubation in the presence or absence of 10% serum. The highly metastatic cell line contained 35% more actin when incubated in the presence of 10% serum, almost completely distributed to the Triton-insoluble cytoskeleton, an effect not seen in the low metastatic cells.     

Molecular characterization of the human platelet integrin GPIIb/IIIa and its constituent glycoproteins

Human platelet plasma membrane glycoproteins IIb (GPIIb) and IIIa (GPIIIa) form a Ca(2+)-dependent heterodimer, the integrin GPIIb/IIIa, which serves as the receptor for fibrinogen and other adhesive proteins at the surface of activated platelets. Below the critical micellar concentration of Triton X100 (TtX), the three glycoproteins do not bind appreciably to TtX and form association products of large size. The size-exclusion chromatographic patterns of GPIIb, GPIIIa and GPIIb/IIIa have been obtained at 0.2% TtX, and the molecular properties of the association products and monomer fractions have been determined by analysis of the detergent bound to the glycoproteins, laser-light scattering, sedimentation velocity, and electron microscopy (TEM). The monomer of the GPIIb-TtX complex was identified by the molecular mass (M) of the glycoprotein moiety (125 +/- 15 kDa), the molecular size (9.5 +/- 1.5 nm x 11 +/- 1.5 nm) and globular shape observed by TEM. It has a molecular mass (M*) of 197 +/- 20 kDa, a sedimentation coefficient s degrees 20* of 5.8 +/- 0.1 S, a Stokes radius R s* of 6.8 +/- 0.4 nm, and a frictional ratio f*/fmin* of 1.7 +/- 0.14. The (GPIIb)n-TtX complexes are disulphide-bonded size-heterogeneous association products of GPIIb, tetramers being the smallest species found. GPIIIa has a greater propensity to self-associate than GPIIb, this tendency being lower below 1 mg GPIIIa/ml, 0.1 mM Ca2+, pH 9.0. The (GPIIIa)n-TtX complexes are noncovalent size-heterogeneous association products of GPIIIa, tetramers being the smallest form observed. The monomer of the GPIIIa-TtX complex was identified by the 103 +/- 15 kDa M determined for the glycoprotein moiety, and the 9 +/- 1.5 nm x 10 +/- 1.5 nm size and globular shape observed by TEM. It has a M* of 136 +/- 15 kDa, a s degrees 20* of 3.9 +/- 0.3 S, a Rs* of 6.4 +/- 0.5 nm, a f*/fmin* of 1.9 +/- 0.3, and, when stored at pH 7.4, has a certain tendency to form filamentous association products (20-70 nm x 2-5 nm), as observed by TEM. The GPIIb/IIIa-TtX complex in 0.2% TtX/0.1 mM Ca2+ elutes as a single monomeric fraction, as deduced from the 210 +/- 15 kDa M determined for its glycoprotein moiety and the 12 +/- 1.5 nm x 14 +/- 1.5 nm size of the globular forms observed by TEM.(ABSTRACT TRUNCATED AT 400 WORDS)     

Isolation of a domain of the plasma membrane in Chinese hamster ovary cells which contains iodinatable, acidic glycoproteins of high molecular weight

A light vesicle fraction, apparently derived from the plasma membrane, was obtained following breakage of Chinese hamster ovary (CHO) cells by means of a fluid pump disrupting device. The final preparation was enriched approx. 40-fold over the homogenate in K+,Na+-stimulated ATPase and phosphodiesterase I, but only approx. 10-fold in 125I specific radioactivity after lactoperoxidase-catalyzed iodination. This preparation was compared with another plasma membrane fraction purified as large sheets via a two-phase centrifugation procedure. Two-dimensional polyacrylamide gel electrophoresis followed by Coomassie blue staining indicated that both fractions were fairly similar in polypeptide composition, although a few consistent differences were evident. However, staining of glycoproteins by the periodic acid-Schiff technique or by overlaying with 125I-labeled concanavalin A showed that the vesicle fraction was highly enriched in groups of high molecular weight, acidic glycoproteins which stain only weakly with Coomassie blue. These glycoproteins also bound 125I-labeled ricin I agglutinin and wheat germ agglutinin. They appear to be the major receptors for wheat germ agglutinin on the CHO cell surface. After surface labeling of cells by the 125I-lactoperoxidase technique, the membrane sheet fraction contained a large number of iodinated polypeptides, whereas labeling in the vesicle fraction was restricted almost entirely to the high molecular weight, acidic glycoproteins. It is proposed that the vesicle fraction constitutes a specific domain of the cell surface which is coated on its exterior by this group of glycoproteins. These components probably mask underlying proteins of the plasma membrane from external labeling.     

Fate of surface proteins of rabbit polymorphonuclear leukocytes during phagocytosis. I. Identification of surface proteins

To study the fate of external membrane proteins during phagocytosis, rabbit peritoneal neutrophils were labeled by enzymatic iodination. Iodine was incorporated into at least 13 proteins ranging in size from approximately 250,000 to 18,000 daltons as judged from autoradiography of gels after SDS-polyacrylamide gel electrophoresis of labeled cells. The major contractile proteins of neutrophils, actin and myosin, were not labeled when intact cells were iodinated but were labeled when homogenates of these cells were iodinated. Nine of the iodinated proteins were released by mild protease treatment of intact cells. A plasma membrane-rich fraction was isolated by density centrifugation. This fraction was enriched at least 10-fold for lactoperoxidase-labeled acid-insoluble proteins. It was enriched to the same extent for the presence of iodinated wheat germ agglutinin that had been bound to intact cells at 4 degrees C before homogenization. Analysis of SDS-polyacrylamide gel electrophoresis revealed that the proteins of this fraction were predominantly of high molecular weight. However, only 8 of the 13 proteins iodinated on intact cells were found in this fraction. The remaining five were enriched in a dense fraction containing nuclei, intact cells, and membranous vesicles, and may represent a specialized segment of the neutrophil cell surface.     

Actin and actin-associated proteins of rabbit liver cell

Rabbit liver actin and its associated proteins were prepared and their properties were studied. Liver cells were isolated from excised rabbit liver after perfusion in situ with calcium-free Lock's solution. Dried powder of acetone-treated liver cells was extracted with a buffer previously used to extract actin from skeletal muscle. The liver actin was recovered by adding skeletal myosin to trap actin as actomyosin and the resulting complex was purified by centrifugation. The actin was then dissociated from myosin by adding MgATP and was purified by centrifugation. This fraction showed the characteristic properties of F-actin and was composed of 42K, 53K, and 61K proteins. Further fractionation of these proteins into three components was carried out by centrifugation, DNase-1 affinity chromatography, and preparative gel electrophoresis. The 42K protein proved to be actin since it activated the myosin Mg2+-ATPase activity, interacted with DNase-1, and had a very similar amino acid composition to skeletal muscle actin. In these experiments, binding affinity among these proteins was apparent. Analysis of subcellular fractions combined with the above results indicated that the liver cell 53K and 61K proteins were not soluble fraction components in the cytosol. The physicochemical properties of 53K and 61K proteins were compared with those of gizzard desmin, a typical intermediate filament protein.     

Identification of actin-binding protein as the protein linking the membrane skeleton to glycoproteins on platelet plasma membranes

Platelets have previously been shown to contain a membrane skeleton that is composed of actin filaments, actin-binding protein, and three membrane glycoproteins (GP), GP Ib, GP Ia, and a minor glycoprotein of Mr = 250,000. The present study was designed to determine how the membrane glycoproteins were linked to actin filaments. Unstimulated platelets were lysed with Triton X-100, and the membrane skeleton was isolated on sucrose density gradients or by high-speed centrifugation. The association of the membrane glycoproteins with the actin filaments was disrupted when actin-binding protein was hydrolyzed by activity of the Ca2+-dependent protease, which was active in platelet lysates upon addition of Ca2+ in the absence of leupeptin. Similarly, activation of the Ca2+-dependent protease in intact platelets by the addition of a platelet agonist also caused the membrane glycoproteins to dissociate from the membrane skeleton. Affinity-purified actin-binding protein antibodies immunoprecipitated the membrane glycoproteins from platelet lysates in which actin filaments had been removed by DNase I-induced depolymerization and high-speed centrifugation. These results demonstrate that actin-binding protein links actin filaments of the platelet membrane skeleton to three plasma membrane glycoproteins and that filaments are released from their attachment site when actin-binding protein is hydrolyzed by the Ca2+-dependent protease within intact platelets during platelet activation.     

The Proteins in Mung Bean Mitochondria

The mung bean mitochondria were obtained by sucrose cushion centrifugation in quite pure form. The outer membrane and the mitoplast were separated by digitonin treatment followed by sucrose density gradient centrifugation. SDS gradient polyacrylamide gel electrophoresis showed that the 42–44 KD protein in mung bean mitochondria was located in mitoplast rather than in the outer membrane. Co-electrophoresis with rabbit muscle actin showed that the rabbit muscle actin coincided with the 44 KD band in plant mitochondria. These results suggest that one of the motive force of the swelling and shrinking of the mitochondria may be generated from the mitochondria themselves.     

Solubilization and enrichment of boar sperm membrane proteins

Boar sperm membranes are rather resistent to the solubilizing effect of some detergents. Deoxycholate, an ionic detergent, was efficient in solubilizing sperm proteins but some nonionic detergents like Triton X-100 displayed relatively poor capacity in rendering membrane proteins soluble. This may be due to sperm proteins being attached to submembraneous structures through bonds involving divalent cations, since mixtures of Triton X-100 and ethylenediamine tetraacetic acid (EDTA) were almost as efficient as deoxycholate in solubilizing membrane proteins. Since intact spermatozoa were directly treated with detergents the solubilized proteins comprised a mixture of intracellular and membrane components. To enrich for membrane proteins, affinity chromatography on columns containing different lectins was carried out. SDS polyacryiamide gel electrophoresis of sperm glycoproteins desorbed from the various lectin columns demonstrated that each lectin bound a unique set of components although most glycoproteins were recovered from two or more columns. Columns containing Lens culinaris hemagglutinin yielded more sperm glycoproteins than any of the other lectin columns examined. The predominant amount of the sperm proteins recovered from the Lens culinaris lectin column was membrane derived, as the majority of the proteins were integrated into liposomes. It is concluded that sperm membrane proteins are efficiently solubilized by detergent in the presence of a chelator and that most of the membrane glycoproteins can easily be enriched by affinity chromatography on a lectin column. Proteins obtained in this way should serve as excellent starting material for the isolation of individual sperm membrane proteins.     

Characterization of the multiple conformational States of free monomeric and trimeric human immunodeficiency virus envelope glycoproteins after fixation by cross-linker

The human immunodeficiency virus type 1 (HIV-1) gp120 exterior and gp41 transmembrane envelope glycoproteins assemble into trimers on the virus surface that represent potential targets for antibodies. Potent neutralizing antibodies bind the monomeric gp120 glycoprotein with small changes in entropy, whereas unusually large decreases in entropy accompany gp120 binding by soluble CD4 and less potent neutralizing antibodies. The high degree of conformational flexibility in the free gp120 molecule implied by these observations has been suggested to contribute to masking the trimer from antibodies that recognize the gp120 receptor-binding regions. Here we use cross-linking and recognition by antibodies to investigate the conformational states of gp120 monomers and soluble and cell surface forms of the trimeric HIV-1 envelope glycoproteins. The fraction of monomeric and trimeric envelope glycoproteins able to be recognized after fixation was inversely related to the entropic changes associated with ligand binding. In addition, fixation apparently limited the access of antibodies to the V3 loop and gp41-interactive surface of gp120 only in the context of trimeric envelope glycoproteins. The results support a model in which the unliganded monomeric and trimeric HIV-1 envelope glycoproteins sample several different conformations. Depletion of particular fixed conformations by antibodies allowed characterization of the relationships among the conformational states. Potent neutralizing antibodies recognize the greatest number of conformations and therefore can bind the virion envelope glycoproteins more rapidly and completely than weakly neutralizing antibodies. Thus, the conformational flexibility of the HIV-1 envelope glycoproteins creates thermodynamic and kinetic barriers to neutralization by antibodies directed against the receptor-binding regions of gp120.     

The Glycoprotein Composition of Peripheral Nervous System Myelin Subfractions

Two fractions were isolated by continuous density gradient centrifugation from total particulate matter of rabbit sciatic nerves: a minor fraction, B, consisting of small-sized membrane fragments and a major fraction, C, of characteristic multilayered myelin figures, with maxima at 0.33 and 0.58 M-sucrose, respectively. In comparison with C, fraction B was enriched in CNPase and alkaline phosphatase activities and the P0, 23K and Z proteins, but was virtually devoid of basic protein. The glycoprotein composition of all fractions was examined with four fluorescein isothiocyanate-labelled lectins (WGA, Con A, RCA-60, U.E.). These revealed the presence of six glycoproteins in all fractions with similar lectin binding capacities and molecular weights ranging from 35,500 to 16,000, of which P0 was the predominant component. Material found on the heavy side of fraction C was characterized by the presence of a multitude of glycoproteins which bound variable proportions of the four different lectins, suggesting substantial variations in their carbohydrate moieties. Their absence from the central portion of fraction C points to a location other than that of compact PNS myelin.     

Monomeric Intermediates Formed by Vesiculovirus Glycoprotein during Its Low-pH-induced Structural Transition

Vesiculoviruses enter cells by membrane fusion, driven by a large, low-pH-induced, conformational change in the fusion glycoprotein (G) that involves transition from a trimeric pre-fusion to a trimeric post-fusion state. G is the model of class III fusion glycoproteins which also includes the fusion glycoproteins of herpesviruses (gB) and baculoviruses (gp64). Class III fusion proteins combine features of the previously characterized class I and class II fusion proteins. In this review, we first present and discuss the data that indicate that the Vesiculovirus G structural transition proceeds through monomeric intermediates. Then, we focus on a recently determined crystal structure of the Chandipura virus G ectodomain that contained two monomeric intermediate conformations of the glycoprotein, revealing the chronological order of the structural changes in the protein and offering a detailed pathway for the conformational change, in agreement with electron microscopy data. In the crystal, the intermediates were associated through their fusion domain in an antiparallel manner to form an intermolecular β-sheet. Mutagenesis indicated that this interface is functionally relevant. All those structural data challenge the current model proposed for viral membrane fusion. Therefore, we wonder if this mode of operating is specific to Vesiculovirus G and discuss data indicating that class II fusion glycoproteins are monomeric when they interact with the target membrane but also crystal structures suggesting the existence of non-trimeric intermediates for influenza hemagglutinin which is the prototype of class I fusion proteins.     

Two-dimensional polyacrylamide-gel electrophoresis of the proteins and glycoproteins of purified human platelet surface and intracellular membranes.

By using highly purified surface and intracellular membrane fractions prepared from human platelets by free-flow electrophoresis, the polypeptide and glycopeptides of these membranes have been characterized by high-resolution gel electrophoresis under reducing and non-reducing conditions. Silver staining and a variety of glycoprotein-staining procedures have been applied to identify the major components. The principal finding was the clear disparity between the distribution patterns for these two membrane fractions. There are proportionately more low-Mr acidic components present in the intracellular membrane than in the surface-derived membrane. Of the major platelet surface glycoproteins GPIb, IIb, IIIa and IIIb (or IV) well expressed in the surface membrane only, GPIIb and IIIa appear as trace components in the intracellular membrane. The cytoskeleton proteins, actin, myosin, tropomyosin, actin-binding protein and alpha-actinin are prominent features of the surface membrane and essentially absent from the intracellular membrane. Neuraminidase treatment at the whole-cell level, before homogenization, which is an essential requirement for good resolution of the two membrane subfractions, modifies a number of the glycoprotein subunits with respect to their pI characteristics, suggesting much molecular micro-heterogeneity with respect to sialic acid content. A comparison of the staining characteristics of the major glycoproteins with periodic acid/Schiff's reagent and concanavalin A/peroxidase detection and a combined procedure revealed significant differences in associated carbohydrate structures, and the major concanavalin A-binding component was shown to be GPIIIa. These observations are discussed in the context of functional activities of both membrane systems in the physiological behaviour of the platelet.