Low-affinity Ca(2+)-binding sites versus Zn(2+)-binding sites in histidine-rich Ca(2+)-binding protein of skeletal muscle sarcoplasmic reticulum.

Histidine-rich Ca(2+)-binding protein (HRC) is a 170 kDa protein that can be identified in the isolated sarcoplasmic reticulum from rabbit skeletal muscle by its ability to bind [125I]low-density lipoprotein on blots after SDS-PAGE and that appears to be bound to the junctional membrane through calcium bridges. Molecular cDNA cloning of this protein predicts the existence of a Ca(2+)-binding domain and of a distinct heavy-metal binding domain at the cystein-rich COOH-terminus. Here we demonstrate, using radioactive ligand blot techniques, that HRC protein binds 45Ca at low affinity, as well as being able to bind 65Zn, but at different sites, that are largely inhibitable by prior reductive alkylation of the protein. In contrast to Ca(2+)-binding protein calsequestrin not having detectable 65Zn-binding sites, HRC protein bound selectively to immobilized Zn2+ on IDA-agarose affinity columns. Our results also indicate that rabbit and human 140 kDa HRC protein have common properties.     

Interaction of laminin with the plasma membrane components of ascitic Zajdela hepatoma cells

The laminin affinity chromatography was used for isolating laminin-binding proteins from the plasma membrane of Zajdela hepatoma cells synthesizing laminin. These were components with mol. weights about 80, 67, 60, 55, 52, 48 and 43 kDa. The isolation of laminin integrin receptors from plasma membranes of Zajdela hepatoma cells in the presence of MnCl2 detected only a protein with mol. weight about 80 kDa in EDTA-elution conditions. This protein was identified by mass spectrometry method as the 78 kDa glucose-regulated protein precursor (GRP78). It belongs to the family of 70 kDa heat shock proteins, recently GRP78 was reported to be localized on the surface of different cell types, including hepatocytes.     

Isolation of a laminin-binding protein from muscle cell membranes

Skeletal muscle myofibers are each ensheathed by a continuous basal lamina consisting predominantly of type IV collagen, laminin and heparan sulfate proteoglycan. In order to identify laminin-binding components in the muscle cell surface, plasma membranes from mouse thigh muscle and from rat L6 myoblasts were separated by polyacrylamide gel electrophoresis and transferred to nitrocellulose paper by electroblotting. Incubation of the transferred samples with ¹²⁵I-labelled laminin revealed a prominent band of approximate mol. wt. 68 000. A protein of this mol. wt. was isolated by affinity chromatography of muscle cell plasma membranes on laminin-Sepharose. The hydrophobic protein has an apparent mol. wt. of 68 000 and has a high content of serine, glycine and acidic amino acids. After detergent solubilization the purified protein binds to laminin-coated Sepharose beads at a higher rate than to beads coated with either fibronectin or collagen types I and IV. The interaction of the protein, called LB 68, with laminin was also studied after incorporation into synthetic lecithin vesicles. While detergent-solubilized LB 68 bound to ¹²⁵I-labeled laminin only at lower than physiological ionic strength, liposome-incorporated LB 68 bound to laminin in the absence of detergents under physiological conditions. We propose that this protein is involved in the interaction of myoblasts with laminin substrates and thus may participate in the anchorage of the basal lamina in the plasmalemma of myotubes.     

Kinetics of receptor-mediated binding of tropoelastin to ligament fibroblasts

Soluble 125I-labeled tropoelastin bound to confluent cultures of bovine ligamentum nuchae fibroblasts and to fibroblast plasma membrane preparations in a time-dependent, saturable, and reversible manner. Scatchard analysis indicates that there are approximately 2 X 10(6) binding sites/cell with a binding efficiency (Kd) of 8 X 10(-9) M. Binding of tropoelastin to cells and membranes reached equilibrium by 90 min and was reversible with 50% of specifically bound material released by 40 min. Specific binding of tropoelastin to cells pre-treated with dilute trypsin solutions was reduced significantly when compared with controls. Four polypeptides of estimated molecular masses of 67, 61, 55, and 43 kDa were obtained from detergent extracts of plasma membranes by elution affinity chromatography on elastin-Affi-Gel. Our findings establish that elastin-specific binding proteins displaying characteristics of a true receptor are present on the surface of elastin-producing cells.     

Isolation of the haemopexin-haem receptor from pig liver cells

Isolated pig liver plasma membranes interact specifically with the haemopexin-haem complex (Kd 4.4 X 10(-7) M). Affinity chromatography was used to isolate a membrane component which binds this complex with high affinity. Pig serum haemopexin was first isolated by affinity chromatography on haemin-Sepharose followed by HPLC gel filtration. Liver membranes solubilized with Triton X-100 were incubated with haemin-Sepharose saturated with haemopexin, and as a control, with affinity gel lacking haemopexin. SDS-poly-acrylamide gel electrophoresis of the eluted protein indicated that from the haemin-Sepharose emerglow-molecular-mass haemin-binding proteins whereas the eluate from haemopexin-haemin-Sepharose contained an additional 71 kDa protein, which did not bind free haemin. This protein appears to represent the haemopexin-haem receptor or a part of it. Haem from the haemopexin complex, as also free haemin, was accepted by a binder in the plasma membrane, which in gel filtration behaved like an 80 kDa molecule. This component probably represents a second functional subunit of the haemopexin-haem receptor.     

Biosynthesis of glycerolipids by hepatoma and liver microsomes. I. Fatty acyl-CoA ligase and acyl-CoA:sn-glycerol-3-phosphate acyltransferase

Highly purified plasma membranes of calf thymocytes were fractionated by means of affinity chromatography on ouabain-Sepharose. By the method used two subfractions were obtained, one eluting freely from the affinity gel (MF1oua) and a second specifically retained by matrix-bound ouabain (MF2oua), with a total recovery of 95 per cent. Fractionation required the binding of matrix-bound ouabain to its plasma membrane receptor, i.e. (Na+ + K+)-ATPase. Increasing the temperature and binding time did not significantly alter the fractionation of plasma membranes into the two subfractions. Both plasma membrane subfractions separated by ouabain-Sepharose were of plasma membrane origin, as revealed by the identical specific activities of several membrane bound enzymes, gamma-glutamyl transpeptidase, alkaline phosphatase and Mg2+-ATPase in unseparated plasma membranes and in both subfractions, and by the identical amounts of the cytoskeletal protein actin in unseparated plasma membranes and subfractions. The plasma membrane subfractions MF1oua and MF2oua showed different structural and functional properties. In SDS-polyacrylamide gel electrophoresis polypeptides of 170, 150, 110, 94, 39, and 30 kDa were several-fold enriched in the adherent fraction, MF2oua. The phospholipid fatty acid composition of the plasma membrane subfractions proved to be different, as well. MF2oua contained significantly higher amounts of saturated fatty acids as compared to MF1oua. The specific activities of (Na+ + K+)-ATPase, Ca2+-ATPase and lysolecithin acyltransferase were highly enriched in the adherent fraction MF2oua, as compared to MF1oua. The data suggest that by the means of affinity chromatography on ouabain-Sepharose plasma membrane domains of the lymphocyte plasma membrane can be isolated, most probably implicated in the initiation of lymphocyte activation.     

Two cell surface proteins bind the sponge Microciona prolifera aggregation factor

Two extracellular matrix cell surface proteins which bind the proteoglycan-like aggregation factor from the marine sponge Microciona prolifera (MAF) and which may function as physiological receptors for MAF were identified and characterized for the first time. By probing nitrocellulose blots of nonreducing sodium dodecyl sulfate gels containing whole sponge cell protein with iodinated MAF, a 210- and a 68-kDa protein, which have native molecular masses of approximately 200-400 and 70 kDa, were identified. MAF binding to blots is species-specific. It is also sensitive to reduction and is completely abolished by pretreatment of live cells with proteases, as was cellular aggregation, indicating that the 210- and 68-kDa proteins may be located on the cell surface. The additional observations that the 68 kDa is an endoglycosidase F-sensitive glycoprotein and that antisera against whole sponge cells or membranes can immunoprecipitate the 210 kDa when prebound to intact cells are consistent with a cell surface location. Both proteins can be isolated from sponge cell membranes and from the sponge skeleton (insoluble extracellular matrix), but the 210-kDa MAF-binding protein can also be found in the soluble extracellular matrix (buffer washes of cells and skeleton) as well. A third MAF-binding protein of molecular mass 95 kDa was also found in the sponge extracellular matrix but rarely on cells. Both of the cell-associated 210- and 68-kDa proteins are nonintegral membrane proteins, based on Triton X-114 phase separation, flotation of liposomes containing sponge membrane lysates, and their extraction from membranes by buffer washes. Both proteins bind MAF affinity resins, indicating that they each exhibit a moderate affinity for MAF under native conditions. They can also be separated from each other and from the bulk of the protein in an octylpolyoxyethylene extract of membranes by fast protein liquid chromatography Mono Q anion exchange chromatography, as assessed by native dot blot and denaturing Western blot assays. Although neither protein bound to heparin, gelatin, hexosamine, or uronic acid-Sepharose resins, their affinity for an invertebrate proteoglycan, their roles in sponge cell adhesion, and their peripheral membrane protein natures suggest that they may represent early invertebrate analogs of cell-associated vertebrate extracellular matrix adhesion proteins, such as fibronectin or vitronectin, or else an entirely novel set of cell adhesion molecules.     

Isolation and partial characterization of plasma membrane fatty acid binding proteins from myocardium and adipose tissue and their relationship to analogous proteins in liver and gut

We describe a general method for isolating a class of 40 kDa plasma membrane fatty acid binding proteins which have been identified previously only in rat liver and jejunum. Proteins extracted with 2 M salt from rat adipocyte and cardiac myocyte plasma membranes were subjected to preparative isoelectric focusing. Fractions with pI's greater than or equal to 9.0 were further purified by oleate-agarose affinity chromatography and HPLC. Each tissue yielded a single 40 kDa protein which co-chromatographed with [3H]-oleate on gel permeation HPLC, and reacted on Western blots with an antibody to the corresponding hepatic membrane protein. Related plasma membrane fatty acid binding proteins have now been isolated from each of the major sites of fatty acid transport.     

A specific insulin receptor and tyrosine kinase activity in the membranes of Neurospora crassa

Cells of the wall-less ("slime") strain of Neurospora crassa possess specific high affinity insulin binding sites on their cell surface. 125I-labeled bound insulin was not displaced from these cells by insulin-like growth factor II (IGF-II), and was only weakly displaced by IGF-I and proinsulin. Cross-linking of 125I-labeled insulin with N. crassa cells using disuccinimidyl suberate resulted in the labeling of a single band of ca. 67 kDa m.w. on a polyacrylamide gel. Two proteins of ca. 66 and 59 kDa m.w. were purified from detergent solubilized plasma membrane preparations by passage over an insulin-agarose affinity matrix. Antibodies against an autophosphorylation site on the human and Drosophila insulin receptors (anti P2) immunoprecipitated a single phosphoprotein of ca. 50 kDa m.w. from detergent solubilized plasma membranes, which possessed protein tyrosine kinase activity when histone H2 was used as substrate.     

Identification of laminin receptor in gingival tissue and its interaction with tooth cementum laminin.

1. A gingival epithelial cell surface receptor for laminin was isolated from bovine gingival tissue by affinity chromatography on laminin. 2. The protein bound by the affinity matrix from octylglucoside extracts of gingival membrane preparation eluted from the column with the cation-free buffer containing EDTA, and exhibited a mol. wt of 67 kDa. 3. The 67 kDa protein following radioiodination was incorporated into liposomes, which showed a specific affinity towards laminin-coated surfaces, as well as to the tooth cementum. 4. The results provide for the first time evidence for the existence of a gingival cell surface laminin receptor, and indicate that the maintenance of the cemento-epithelial junction involves the interaction between the cementum laminin and its receptor on gingival epithelium.     

Human microvascular endothelial cells use beta 1 and beta 3 integrin receptor complexes to attach to laminin

Microvascular endothelial cells (MEC) use a set of surface receptors to adhere not only to the vascular basement membrane but, during angiogenic stimulation, to the interstitium. We examined how cultured human MEC interact with laminin-rich basement membranes. By using a panel of monoclonal antibodies, we found that MEC cells express a number of integrin-related receptor complexes, including alpha 1 beta 1, alpha 2 beta 1, alpha 3 beta 1, alpha 5 beta 1, alpha 6 beta 1, alpha V beta 3. Attachment to laminin, a major adhesive protein in basement membranes, was studied in detail. Blocking monoclonal antibodies specific to different integrin receptor complexes showed that the alpha 6 beta 1 complex was important for MEC adhesion to laminin. In addition, blocking antibody also implicated the vitronectin receptor (alpha V beta 3) in laminin adhesion. We used ligand affinity chromatography of detergent-solubilized receptor complexes to further define receptor specificity. On laminin-Sepharose columns, we identified several integrin receptor complexes whose affinity for the ligand was dependent on the type of divalent cation present. Several beta 1 complexes, including alpha 1 beta 1, alpha 2 beta 1, and alpha 6 beta 1 bound strongly to laminin. In agreement with the antibody blocking experiments, alpha V beta 3 was found to bind well to laminin. However, unlike binding to its other ligands (e.g., vitronectin, fibrinogen, von Willebrand factor), alpha V beta 3 interaction with laminin did not appear to be Arg-Gly-Asp (RGD) sensitive. Finally, immunofluorescent staining demonstrated both beta 1 and beta 3 complexes in vinculin-positive focal adhesion plaques on the basal surface of MEC adhering to laminin-coated substrates. The results indicate that both these subfamilies of integrin heterodimers are involved in promoting MEC adhesion to laminin and the vascular basement membrane.     

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.     

Zinc distribution and zinc-binding forms in Phragmites australis under zinc pollution

The influence of zinc (Zn) on physiological and biochemical parameters was studied to elucidate the mechanism of Zn resistance in Phragmites australis. Zn concentrations in roots, stems and leaves increased with exogenous Zn concentration, while Zn content in roots was much higher than in shoots. X-ray microanalysis was used to reveal compartments in which Zn accumulated in root cortex. Zinc concentrations followed a gradient with the sequence: intercellular space>cell wall >vacuole >cytoplasm, indicating that most Zn was immobilized in the apoplast or sequestered into the vacuolar lumen. Sequential extraction of various Zn chelates revealed that the ratio of Zn extracted with different extraction media was markedly different. Ethanol, HAc (acetic acid) and NaCl-extractable Zn were dominant in both roots and leaves of P. australis. Zn-binding protein fractions were found in the roots and leaves after gel filtration chromatography, among which a polypeptide with an apparent molecular mass of 14kDa bound Zn most effectively. Two newly synthesized polypeptides of 58 and 45kDa appeared under Zn pollution, whereas a prominent fraction of 72kDa disappeared. The involvement of Zn distribution in plant tissues, subcellular compartments and chelates and Zn-inducing proteins in the acclimation mechanism of P. australis to Zn pollution is discussed.     

Calmodulin-binding proteins in granule and plasma membranes from bovine chromaffin cells

Calmodulin-binding proteins in chromaffin granule membrane and chromaffin cell plasma membranes have been investigated and compared. Chromaffin granules were purified by centrifugation over a 1.7 M sucrose layer. Plasma membranes were obtained in a highly purified form by differential and isopycnic centrifugation. Enzymatic determinations of 5'-nucleotidase, a generally accepted plasma membrane marker, showed a 40-50-fold enrichment as compared to the cell homogenate. Marker enzyme studies demonstrated only minimal contamination by other subcellular organelles. After solubilization with Triton X-100, calmodulin-binding proteins were isolated from chromaffin granule membranes and plasma membranes by affinity chromatography on a calmodulin/Sepharose 4B column. On two-dimensional polyacrylamide gelelectrophoresis a prominent protein (Mr = 65,000, pI ranging from 5.1 to 6) consisting of multiple spots, was present in the calmodulin-binding fraction from chromaffin granule membranes as well as from plasma membranes. Besides this 65 kDa protein both fractions had at least four groups of proteins in common. Also, proteins typical for either preparation were observed. In the calmodulin-binding protein preparations from chromaffin granule membranes a prominent spot with Mr = 80,000 and a pH ranging from 5.0 to 5.7 was present. This protein was enzymatically and immunologically identified as dopamine-beta-monooxygenase.     

Characterization and purification of the hyaluronan-receptor on liver endothelial cells.

In order to characterize the proteins on liver endothelial cells that bind hyaluronan (HYA), liver endothelial cells were surface-iodinated with 125I, solubilized by Triton X-100 and passed through a column containing HYA coupled to agarose. The column was washed and eluted with HYA-oligosaccharides. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the eluted material, followed by autoradiography, showed a major band with a molecular mass of 100 kDa, that upon reduction gave major bands of 20 and 35 kDa, and minor doublet bands at 60 and 80 kDa. Two-dimensional electrophoresis of liver endothelial cell membrane proteins revealed that the 100 kDa protein has a pI of 6.6-6.8. The protein was purified by preparative SDS-PAGE of liver endothelial cell membrane proteins. The 100 kDa protein was excised from the gel and used for immunization of rabbits. Antiserum from immunized rabbits specifically recognized only the 100 kDa protein on immunoblots of liver endothelial cell membrane proteins separated by SDS-PAGE. The binding of 3H-HYA to liver endothelial cells and liver endothelial cell membranes could be specifically inhibited by Fab-fragments of the antibodies. When we tried to isolate the receptor in large scale by affinity chromatography of proteins from purified liver endothelial cell membranes, the 100 kDa protein could often not be detected on immunoblots or by silver staining following SDS-PAGE of the eluted material. Instead, proteins with molecular masses of 55 and 15 kDa were detected, but the antibodies reacted specifically with these proteins. Thus the 100 kDa protein is apparently susceptible to cleavage into distinct subcomponents.     

Equilibrium dialysis measurements of the Ca2+-binding properties of recombinant radish vacuolar Ca2+-binding protein expressed in Escherichia coli

Vacuoles of radish (Raphanus sativus) contained a Ca2+-binding protein (RVCaB) of 43 kDa. We investigated the Ca2+-binding properties of the protein. RVCaB was expressed in Escherichia coli and was purified from an extract by ion-exchange chromatography, nitrocellulose membrane filtration, and gel-filtration column chromatography. Ca2+-binding properties of the recombinant protein were examined by equilibrium dialysis with 45Ca2+ and small dialysis buttons. The protein was estimated to bind 19Ca2+ ions per molecule with a Kd for Ca2+ of 3.4 mM. Ca2+ was bound to the protein even in the presence of high concentrations of Mg2+ or K+. The results suggested that the protein bound Ca2+ with high ion selectivity, high capacity, and low affinity.     

Purification and properties of a membrane-bound insulin binding protein, a putative receptor, from Neurospora crassa.

The protein that is responsible for specific, high-affinity binding of insulin to the surface of Neurospora crassa cells has been purified to homogeneity. The insulin binding activity of solubilized plasma membranes resembled that of intact cells with regard to affinity of binding, specificity for mammalian insulins, and amount of insulin bound per cell. Insulin binding activity was purified from Triton X-100 solubilized membranes in two steps: FPLC on a MonoQ HR5/5 column; and affinity chromatography on insulin-agarose. The pure material migrated as a single band of ca. 66 kDa on SDS gels, pI = 7.4 by isoelectric focusing. The protein bound 5.34 pmol of insulin/micrograms, or 35% of that expected for univalent binding. Cross-linking of 125I-insulin to pure protein or to solubilized membranes revealed a single labeled band of 67-70 kDa on SDS gels. In nonreducing native gels, two labeled bands of ca. 55 and 110 kDa were produced after cross-linking, and two bands of similar molecular weight bound iodinated insulin after transfer to nitrocellulose filters. These may correspond to active monomer and dimer forms. The pure protein possessed no protein kinase activity against itself, or against exogenous substrates (histone H2, casein, or the synthetic peptide Glu80-Tyr20), and possessed no detectable phosphorylated amino acids. It is suggested, however, that this 66-kDa protein is the "receptor" that mediates insulin-induced downstream metabolic effects.     

Glycoproteins in the whorls of membrane produced by oligodendroglia in culture

Two glycoproteins of 99 kDa and 77 kDa which exhibit intense binding to wheat germ agglutinin have been purified from the whorls of membrane produced by oligodendroglia in culture. The whorls of membrane were isolated by gradient centrifugation from purified bovine oligodendroglia maintained in culture. The two glycoproteins were solubilized from the membranes using a non-ionic detergent and purified by Sephadex LH-60 chromatography, wheat germ agglutinin affinity chromatography, and SDS-polyacrylamide pore gradient gel electrophoresis. HPLC peptide mapping of the 99-kDa and 77-kDa glycoproteins revealed structural differences between the two proteins. Peptide mapping suggested that the 99-kDa glycoprotein from the whorls of membrane may be homologous to that from the plasma membranes. The 77-kDa glycoproteins from both sets of membrane may also be structurally related. Lectin binding studies showed that both glycoproteins from the whorls of membrane bound to wheat germ agglutinin, succinylated wheat germ agglutinin, concanavalin A, and lentil lectin, indicating the presence of high mannose and hybrid type oligosaccharide side-chains.     

Adenosine transporters in chromaffin cells: subcellular distribution and characterization

Adenosine transporters in freshly isolated and cultured chromaffin cells were quantified by the [3H]dipyridamole binding technique, showing a maximal bound capacity of 0.4 +/- 0.05 pmol/10(6) cells (240,000 +/- 20,000 transporters by cell). Scatchard analysis showed a similar affinity for [3H]dipyridamole in isolated cells and subcellular fractions (Kd = 5 +/- 0.6 nM). For enriched plasma membrane preparations and chromaffin granule membranes, the maximal binding capacities were also very similar, 2.3 +/- 0.3 and 1.8 +/- 0.4 pmol/mg protein, respectively. When [3H]nitrobenzylthioinosine was employed as a radioligand, the maximal bound capacity in cultured chromaffin cells was 0.053 +/- 0.004 pmol/10(6) cells (32,000 +/- 3000 transporters per cell) with a high affinity constant (Kd = 0.25 +/- 0.03 nM); similar values were obtained in all subcellular fractions (Kd = 0.1 +/- 0.01). Also, plasma and chromaffin granule membranes showed similar maximal binding values (0.4 +/- 0.06 pmol/mg protein). Photoincorporation studies with [3H]nitrobenzylthioinosine into plasma membrane polypeptides showed the presence of three molecular species of 115 +/- 10; 58 +/- 6 and 42 +/- 5 kDa. Chromaffin granule membranes showed only the 105 +/- 9 and 51 +/- 4 molecular species.     

Characterization of Bufo arenarum oocyte plasma membrane proteins that interact with sperm

Sperm-oocyte plasma membrane interaction is an essential step in fertilization. In amphibians, the molecules involved have not been identified. Our aim was to detect and characterize oocyte molecules with binding affinity for sperm. We isolated plasma membranes free from vitelline envelope and yolk proteins from surface-biotinylated Bufo arenarum oocytes. Using binding assays we detected a biotinylated 100 kDa plasma membrane protein that consistently bound to sperm. Chromatographic studies confirmed the 100 kDa protein and detected two additional oocyte molecules of 30 and 70 kDa with affinity for sperm. Competition studies with an integrin-interacting peptide and cross-reaction with an anti-HSP70 antibody suggested that the 100 and 70 kDa proteins are members of the integrin family and HSP70, respectively. MS/MS analysis suggested extra candidates for a role in this step of fertilization. In conclusion, we provide evidence for the involvement of several proteins, including integrins and HSP70, in B. arenarum sperm-oocyte plasma membrane interactions.