Identification of membrane protein associated with sucrose transport into cells of developing soybean cotyledons

The photolyzable sucrose derivative 6′-deoxy-6′-(4-azido-2-hydroxy)-benzamidosucrose (6′-HABS), competitively inhibited the influx of [¹⁴C] sucrose into protoplasts from developing soybean (Glycine max L. Merr cv Wye) cotyledons. Photolysis of ¹²⁵I-labeled 6′-HABS in the presence of 10 millimolar dithiothreitol and microsomal preparations from developing soybean cotyledons led to label incorporation into a moderately abundant membrane protein with an apparent molecular mass of about 62 kilodalton (kD) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 62 kD protein was partially protected from labeling by the inclusion of 100 millimolar sucrose in the photolysis medium and also by the inclusion of 10 millimolar phenyl α-d-thioglucopyranoside. Glucose, raffinose, or phenyl α-d-3-deoxy-3-fluoroglucopyranoside did not afford even partial protection from labeling. When the photolyzable moiety of 6′-HABS was attached to 6-deoxy-6-aminoglucose and ¹²⁵I labeled, the resulting photoprobe did not label the 62 kD protein above background. The labeled protein at 62 kD is therefore apparently a specific, sucrose binding protein. Sucrose influx into cotlyedons of less than 25 milligrams fresh weight (approximately 10 days after flowering) occurred by passive processes, but metabolically dependent uptake became dominant over the next 5 to 7 days of development. Both the Coomassie staining protein at 62 kD and label incorporation at that position in analysis of membrane proteins appeared concomitant with the onset of active sucrose influx. Polyclonal antibodies to the purified 62 kD protein bound specifically to a protein in the plasmalemma of thin sections prepared from cotyledons and density stained with colloidal gold-protein A. The results suggest that the 62 kD membrane protein is associated with sucrose transport and may be the plasmalemma sucrose transporter.     

A collagen-binding glycoprotein from bovine platelets is identical to propolypeptide of von Willebrand factor

Several proteins from bovine platelet lysate bound to type I collagen immobilized to the beads of formyl derivatives of cellulose. Among these proteins, a protein of about 100,000 daltons was purified to homogeneity by two additional affinity chromatographies, an organomercurial-agarose and a lentil lectin-agarose. This protein consisted of a single polypeptide chain which contains carbohydrate moiety and many intrapolypeptide disulfide bridges. In addition to platelets, this protein was present in plasma and cultured endothelial cells but not in red blood cells, leukocytes, and smooth muscle cells. Furthermore, it was released from platelets upon stimulation by various agonists. The purified 100-kDa protein was labeled with 125I to quantitate its binding to fibrillar type I collagen. The protein specifically bound to fibrillar collagen with the apparent dissociation constant of 5.6 x 10(-8) M for the high affinity site and 5.5 x 10(-7) M for the low affinity site. Analyses of amino acid sequences of both intact and tryptic fragments of this protein revealed that it had strong homology to the propolypeptide of human von Willebrand factor, which is also known as von Willebrand antigen II. Various properties of this protein listed above also strongly suggest that it was indeed the propolypeptide of bovine von Willebrand factor.     

The radioisotope labelling of thrombocytes using a monoclonal antibody against membrane glycoproteins IIb-IIIa and the measurement of thrombocyte adhesion/aggregation on the substrate surface]

A new method for platelet labeling based on binding of monoclonal antibody to human platelets has been suggested in this study. Monoclonal antibody VM16a against membrane glycoproteins IIb-IIIa was labeled by 125I and then incubated with platelets. About 70% of added antibody was bound when it was used at the concentrations corresponding to the linear part of the concentration curve (0.5 and 1.0 micrograms/ml). Due to high efficiency of binding 125I-VM16a-labeled platelets were used for the measurement of adhesion/aggregation to the substrate in platelet-rich plasma without washing of the free label. Experiments with washed platelets double labeled with 51Cr and 125I-VM 6a showed high correlation between the data obtained with both labels. The method of platelet labeling has been applied for the assessment of drug action on platelet adhesion/aggregation. Measurements were performed in platelet-rich plasma and adhesion/aggregation was stimulated by ADP and analogue of thromboxane A2, U46619. It was shown/that antianginal drug trapidil strongly inhibited and antiatherogenic drug probucol did not affect platelet adhesion/aggregation stimulated by both agonists.     

Identification of a constituent of the junctional feet linking terminal cisternae to transverse tubules in skeletal muscle

This study describes the biochemical composition of junctional feet in skeletal muscle utilizing a fraction of isolated triad junctions. [3H]Ouabain entrapment was employed as a specific marker for T-tubules. The integrity of the triad junction was assayed by the isopycnic density of [3H]ouabain activity (24-30% sucrose for free T-tubules, 38- 42% sucrose for intact triads). Trypsin, chymotrypsin, and pronase all caused separation of T-tubules from terminal cisternae, indicating that the junction is composed as least in part of protein. Trypsin and chymotrypsin hydrolyzed four proteins: the Ca2+ pump, a doublet 325,000, 300,000, and an 80,000 Mr protein. T-tubules which had been labeled covalently with 125I were joined to unlabeled terminal cisternae by treatment with K cacodylate. The reformed triads were separated from free T-tubules and then severed by passage through a French press. When terminal cisternae were separated from T-tubules, some 125I label was transferred from the labeled T-tubules to the unlabeled terminal cisternae. Gel electrophoresis showed that, although T-tubules were originally labeled in a large number of different proteins, only a single protein doublet was significantly labeled in the originally unlabeled terminal cisternae. This protein pair had molecular weights of 325,000 and 300,000 daltons. Transfer of label did not occur to a substantial degree without K cacodylate treatment. We propose that the transfer of 125I label from T-tubules to terminal cisternae during reformation and breakage of the triad junction is a property of the protein which spans the gap between T-tubules and terminal cisternae.     

Identification of a surface protein of the rabbit blood platelet with high affinity for collagen

Platelet membrane components adhering with high affinity to collagen fibers were studied by means of an affinity column in which fibrillar type I collagen was physically immobilized. Intact rabbit platelets in 1 mM EGTA adhered to the column but did not aggregate. Adhesion was dependent on the collagen concentration and on the number of platelets applied. Passage through the column without adhesion did not affect the potential for subsequent platelet binding. Surface-labelled whole platelets were passaged through this column, lysed in Triton and in SDS and labelled components adhering to the collagen were analysed on SDS-polyacrylamide gels. It was found that Triton lysis removed most of the major surface glycoproteins but left the cytoskeleton on the column. Subsequent SDS elution removed the cytoskeletal proteins along with the remaining major surface glycoproteins. The label left on the column could not be eluted with 8 M urea or up to 4 M NaCl. Collagenase digestion of the column collagen released a single surface glycoprotein of Mr 80,000. Limited chymotryptic digestion of the labelled platelets prior to their application to the column did not affect their binding. A radiolabelled band of the same molecular weight (MW) became bound to the collagen following passage of the chymotrypsin-treated platelets. This band was trypsin-sensitive following SDS-polyacrylamide gel electrophoresis (SDS-PAGE). These results, along with other published evidence, suggest that at least one platelet membrane component, expressed on the surface of the unstimulated platelet, binds with high affinity to fibrillar type I collagen and is probably involved in platelet collagen recognition.     

Demonstration of specific binding sites for pituitary adenylate cyclase activating polypeptide (PACAP) in rat astrocytes

The high and low affinity binding sites for PACAP were identified in rat astrocytes using [125I]PACAP27 as the labeled ligand. Scatchard analysis of displacement of the bound tracer by unlabeled PACAP27 indicated the existence of two classes of binding sites, with the dissociation constant (Kd) = 1.22 +/- 0.4 nM, the binding maximal capacity (Bmax) = 821 +/- 218 fmols/mg protein for the high affinity binding site, and Kd = 0.59 +/- 0.06 microM, Bmax = 563 +/- 12 pmols/mg protein for the low affinity binding site, respectively. The specificity of [125I]PACAP27 binding was tested using PACAP38 and peptides structurally related to PACAP, such as VIP, GHRF, PHI, secretin and glucagon. PACAP38 completely displaced the binding of [125I]PACAP27 and Scatchard analysis also indicated the presence of two classes of binding sites with similar Kd and Bmax to those for PACAP27. VIP and GHRF competed with [125I]PACAP27, but to a much lesser extent than unlabeled PACAP27 in binding. Other peptides tested did not displace the binding of [125I]PACAP27 at 10(-6) M.     

Characteristics of collagen-induced fibrinogen binding to human platelets

Polymerized type I calf skin collagen induced a time-dependent specific binding of 125I-fibrinogen to washed human platelets. Binding occurred more rapidly in a shaken rather than in an unstirred system. It was linear in the range 0.05-0.3 microM added fibrinogen and was saturated at higher fibrinogen concentrations (more than 0.8 microM). Scatchard analysis showed a single population of binding sites (16530 +/- 5410 per platelet) with a Kd = 0.53 +/- 0.23 microM. Collagen-induced 125I-fibrinogen binding to platelets was completely inhibited by ADP antagonists such as creatine phosphate/creatine phosphokinase and AMP, and partially inhibited by pretreatment of the platelets with aspirin. With both normal and aspirin-treated platelets a close correlation was observed between the amount of 125I-fibrinogen bound and the extent of dense granule secretion. Our results confirm that fibrinogen becomes bound to platelet surface receptors during collagen-induced platelet aggregation and suggest that secreted ADP is an essential cofactor in this process.     

Effects of prostacyclin analogue (carbacyclin) on the interaction of platelets with different collagen substrates. Inhibition of cAMP increase by collagens type I, III, and IV

We investigated the effects of a stable prostacyclin analogue, carbacyclin, on the interaction of platelets with collagen substrates differing in their ability to activate platelets: human collagens type I, III, IV and V (CI, CIII, CIV and CV), and commercial calf skin collagen type I (CSC). The total adhesion was measured using 51Cr-labelled platelets, and quantitative morphometry of adherent platelets was performed by scanning electron microscopy (SEM). Carbacyclin in the concentrations inducing a 10-fold rise in platelet cAMP did not affect the adhesion of platelets to weak substrates, CV and CSC, but reduced the adhesion to strong substrates, CIV (by 49%) and CI/CIII (by 78%), which stimulated massive spreading and formation of surface-bound aggregates respectively. Carbacyclin inhibited all morphological manifestations of platelet activation associated with adhesion: conversion of native discoid platelets to spherical ones on CSC; massive spreading on CIV; and aggregate formation on CI/CIII. Massive spreading and aggregation on a weak substrate (CSC) stimulated by arachidonic acid and thrombin was also inhibited by carbacyclin. Under the same concentration of agonists aggregation of platelets was more sensitive to the action of carbacyclin, than spreading. Strong collagen substrates CI, CIII and CIV, but not CV and gelatin, inhibited the carbacyclin-induced rise in platelet cAMP.     

Alboaggregin-B: a new platelet agonist that binds to platelet membrane glycoprotein Ib.

A new protein, called alboaggregin-B (AL-B), has been isolated from Trimeresurus albolabris venom by ion-exchange chromatography. It agglutinated platelets without the need for Ca2+ or any other cofactor. The purified protein showed an apparent molecular mass on SDS-PAGE and gel filtration of about 23 kDa under nonreducing conditions. Ristocetin did not alter the binding of AL-B to platelets or affect AL-B-induced platelet agglutination. Agglutinating activity was not dependent on either proteolytic or lectin-like activity in AL-B. Binding analysis showed that AL-B bound to platelets with high affinity (Kd = 13.6 +/- 9.3 nM) at approximately 30,800 +/- 14,300 binding sites per platelet. AL-B inhibited the binding of labeled bovine von Willebrand factor (vWF) to platelets. Monoclonal antibodies against the 45-kDa N-terminal domain of platelet glycoprotein Ib inhibited the binding both of AL-B and of bovine vWF to platelets, and also inhibited platelet agglutination induced by AL-B and bovine vWF. Specific removal of the N-terminal domain of GPIb by treatment of the platelets with elastase or Serratia marcescens protease reduced the binding of labeled AL-B and bovine vWF to platelets and blocked platelet agglutination caused by both agonists. Monoclonal antibodies to glycoprotein IIb/IIIa, to bovine vWF, and to bovine serum albumin did not show any effect on the binding of AL-B to platelets. Our results indicate that the binding domain for AL-B on platelet GPIb is close to or identical with the one for vWF. This new protein may be a very useful tool for studying the interaction between platelets and vWF.     

Labeling of the proteins at the surface of the pancreatic zymogen granule using diazotized [125I]iodosulfanilic acid

Purified pig and rat pancreatic zymogen granules have been covalently labeled with the membrane impermeant agent diazotized [125I]iodosulfanilic acid. Following alkaline lysis, the radioactivity was almost entirely (92%) recovered in a dense protein pellet designated as the 1 M sucrose pellet. The rest (8%) of the label was recovered in the membrane fraction. The specificity of this procedure in labeling the cytoplasmic aspect of the granule is demonstrated by the absence of label from granule content proteins and by the removal of iodinatable proteins following protease treatment of intact granules. No characteristic integral membrane proteins were labeled. In the pig, four major protein bands were labeled in both subfractions at Mr of 15,000, 33,000, 35,000 and 38,000. In the rat, a similar set of protein bands was labeled except for that of 15,000 Mr which was poorly labeled. Due to their location, it is suggested that these proteins may play an important role in the recognition between the granule membrane and the cell membrane and thereby the control of the exocytosis process.     

Affinity labeling of a human platelet membrane protein with 5'-p-fluorosulfonylbenzoyl adenosine. Concomitant inhibition of ADP-induced platelet aggregation and fibrinogen receptor exposure

Incubation of washed human blood platelets with 5'-p-fluorosulfonylbenzoyl [3H]adenosine (FSBA) covalently labels a single polypeptide of Mr = 100,000. Protection by ADP has suggested that an ADP receptor on the platelet surface membrane was modified. The modified cells, unlike native platelets, failed to aggregate in response to ADP (100 microM) and fibrinogen (1 mg/ml). The extent of binding of 125I-fibrinogen and aggregation was inhibited to a degree related to the incorporation of 5'-p-sulfonylbenzoyl adenosine (SBA) into platelets, indicating FSBA could inhibit the exposure of fibrinogen receptors by ADP necessary for aggregation. Incubation of SBA platelets with alpha-chymotrypsin cleaved the covalently labeled polypeptide and concomitantly reversed the inhibition of aggregation and fibrinogen binding. Platelets proteolytically digested by chymotrypsin prior to exposure to FSBA did not require ADP for aggregation and fibrinogen binding. Moreover, subsequent exposure to FSBA did not inhibit aggregation or fibrinogen binding. The affinity reagent FSBA can displace fibrinogen bound to platelets in the presence of ADP, as well as promote the rapid disaggregation of the platelets. The apparent initial pseudo-first order rate constant of dissociation of fibrinogen was linearly proportional to FSBA concentrations. These studies suggest that a single polypeptide can be altered either by ADP-induced conformational changes or proteolysis by chymotrypsin to reveal latent fibrinogen receptors and promote aggregation of platelets after fibrinogen binding.     

Effect of prostacyclin analogue (carbacyclin) on the interaction of platelets with different collagen substrates. Inhibition of camp inccrease by collagens type I, III and IV

We investigated the effects of a stable prostacyclin analogue, carbacylcin, on the interaction of platelets with collagen substrates differing in thier ability to activate platelets: human collagens type I. III, IV and V (CI, CIII, CIV and CV), and commercial calf skin collagen type I (CSC). The total adhension was measured using ⁵¹Cr-labelled platelets, and quantitative morphometry of adherent platelets was performed by scanning electron microscopy (SEM). Carbacyclin in the concentrations inducing a 10-fold rise in platelets cAMP did not effect the adhension of platelets to weak substrates, CV and CSV, but reduced the adhesion to strong substrates, CIV and *by 49%) and CI/CIII (by 78%), which stimulated massive spreading and formation of surface-boud aggregates respectively. Carbacyclin inhibited all morphological manifestations of platelet activation associated with adhension: conversion of native discoid platelets to spherical ones on CSC; massive spreading on CIV; and aggregate formation on CI/CIII. Massive spreading and aggregation on a weak substrate (CSC) stimulated by arachidonic acid and thrombin was also inhibited by carbacyclin. Under the same concentration of angonists aggregation of platelets was more sensitive to the action of carbacyclin, than spreading. Strong collagen substrates CI, CIII and CIV, but not CV and gelatin, inhibited the carbacyclin-induced rise in platelet cAMP.     

Low molecular weight kininogen binds to platelets to modulate thrombin-induced platelet activation

The kininogens, high molecular weight kininogen (HK) and low molecular weight kininogen (LK), are multifunctional, single-gene products that contain bradykinin and identical amino-terminal heavy chains. Studies were performed to determine if LK would bind directly to platelets. 125I-LK specifically bound to gel-filtered platelets in the presence of 50 microM Zn2+. HK effectively competed with 125I-LK for the same binding site (Ki = 27 +/- 9 nM, n = 5). Similarly, the Ki for LK inhibition of 125I-LK binding was 12 +/- 1 nM (n = 3). Albumin, fibrinogen, factor XIII, and kallikrein did not inhibit 125I-LK binding to unstimulated platelets. 125I-LK (66 kDa) was not cleaved upon binding to platelets. The binding of 125I-LK to unstimulated platelets was found to be fully reversible by the addition of a 50 molar excess of unlabeled LK at both 10 and 20 min. LK binding to platelets was saturable with an apparent Kd of 27 +/- 2 nM (mean +/- S.E., n = 9) and 647 +/- 147 binding sites/platelet. Both LK and HK at plasma concentrations inhibited thrombin-induced platelet aggregation. LK and HK at about 5% of plasma concentration also inhibited thrombin-induced secretion of both stirred and unstirred platelets. Both kininogens were found to be noncompetitive inhibitors of proteolytically active thrombin binding to platelets. The kininogens did not inhibit D-phenylalanyl-prolyl-arginine chloromethyl ketone-treated thrombin from binding to platelets. These studies indicated that both kininogens have a region on their heavy chain which allows them to bind to platelets. Further, kininogen binding by its heavy chain modulates thrombin activation of platelets since it prevents proteolytically active thrombin from binding to its receptor.     

Cyclic oxidation-reduction reactions regulate thromboxane A2/prostaglandin H2 receptor number and affinity in human platelet membranes

The radiolabeled thromboxane A2/prostaglandin H2 (TXA2/PGH2) agonist 125I-BOP bound to the TXA2/PGH2 receptor on human platelet membranes. Scatchard analysis showed that pretreatment of platelet membranes with the reducing agent dithiothreitol (DTT) (10 mM) for 10 min decreased maximal 125I-BOP binding (Bmax) from 1.51 +/- 0.11 pmol/mg to 0.51 +/- 0.05 pmol/mg (p = 0.001) and increased the affinity of the remaining binding sites (Kd = 647 +/- 64 pM (untreated), 363 +/- 46 pM (treated), p = 0.006). Prolonged incubation of membranes with DTT (10 mM) for 40 min further reduced the Bmax to 0.23 +/- 0.08 pmol/mg (p = 0.001 from untreated), and the binding affinity remained elevated (Kd = 334 +/- 117 pM, p = 0.035 from untreated). Kinetic analysis of 125I-BOP binding indicated that the apparent increase in binding affinity after DTT treatment was due exclusively to an increase in the rate of ligand-receptor association with no change in dissociation rate. The effects of DTT on 125I-BOP binding were dose-dependent with an EC50 of 8.1 +/- 0.2 mM. DTT inactivation of TXA2/PGH2 receptors was time-dependent with a second order rate constant (k2) of 0.123 M-1 s-1 at 20 degrees C. The platelet membrane 125I-BOP binding site was partially protected from DTT inactivation by prior occupation with the ligand. TXA2/PGH2 receptor protection by I-BOP was dose-dependent and linearly related (r = 0.97, p = 0.002) to the proportion of receptors occupied, but was incomplete since agonist occupation of 89% of the total number of receptors resulted in only a 38% protective effect. Inhibition of 125I-BOP binding after reduction with DTT could be made permanent by addition of the sulfhydryl alkylating agent N-ethylmaleimide (25 mM), but was completely reversed by reoxidation with dithionitrobenzoic acid (DTNB) (5 mM). Oxidation of untreated receptors with DTNB resulted in a 64% increase in 125I-BOP binding sites from 1.65 +/- 0.12 pmol/mg to 2.70 +/- 0.08 pmol/mg (p = 0.013) without affecting binding affinity. DTNB-induced increases in 125I-BOP binding were concentration-dependent with an EC50 of 668 +/- 106 microM and occurred in less than 1 min at 37 degrees C. In the absence of DTT, alkylation of free sulfhydryl groups with N-ethylmaleimide reduced 125I-BOP Bmax in platelet membranes to 0.85 +/- 0.08 pmol/mg (p = 0.003), but did not change the affinity of the remaining receptors. The EC50 for N-ethylmaleimide inactivation of TXA2/PGH2 receptors was 139 +/- 8 mM, and the k2 in time course experiments was 0.067 M-1 s-1 at 20 degrees C.(ABSTRACT TRUNCATED AT 400 WORDS)     

Determination of platelet adhesion to collagen and the associated formation of phosphatidic acid and calcium mobilization

A method was developed to study the adhesion of platelets to fibrillar collagen at 37 degrees C in the absence of aggregation. Human platelets were labeled with [3H]-oleic acid, gel-filtered, and incubated with collagen in the presence of receptor antagonists to thromboxane A2, 5-hydroxytryptamine, and platelet-activating factor, as well as a fibrinogen/fibronectin inhibitor and an ADP-removing system. Those platelets that adhered to collagen were separated from those that did not by filtration through a 10-microns nylon mesh and the extent of platelet adhesion was quantitated by determination of the radioactivity retained by the mesh. The extent of platelet adhesion was proportional to the amount of collagen added up to 100 micrograms/ml and was essentially complete by 1 min. At least 80-90% of the platelets were capable of adhering to collagen. Adhesion was potentiated by the presence of extracellular Mg2+ and this potentiation was inhibited by extracellular Ca2+. Phosphatidic acid increased markedly in those platelets that adhered to collagen and this was associated with increases in cytosolic free Ca2+ levels that could be detected using the fluorescent Ca2+ indicator fura-2.     

Characterization of the binding of human tissue-type plasminogen activator to platelets

Cell surface binding sites for the constituent proteins of the fibrinolytic system may play a role in the localization and regulation of fibrinolysis. In the present study, specific binding of recombinant human tissue-type plasminogen activator (rt-PA) to human blood platelets was identified and characterized. 125I-labeled rt-PA was found to bind specifically, saturably, and reversibly to the surface of gel-filtered platelets, reaching equilibrium within 5 min at 22 degrees C. Scatchard analysis revealed a single class of binding sites. Unstimulated platelets bound 120,000 +/- 24,000 (mean +/- S.D.) molecules/platelet with an apparent Kd of 340 +/- 25 nM, whereas thrombin-stimulated platelets bound 290,000 +/- 32,000 molecules/platelet with an apparent Kd of 800 +/- 60 nM. Binding of 0.1 microM 125I-rt-PA was greater than 90% reversible by a 50-fold excess of unlabeled rt-PA. Binding was not inhibited by fibrinogen or single chain urokinase-type plasminogen activator, but plasminogen partially competed for binding of 125I-rt-PA to platelets (up to 40% displacement). These findings indicate that the platelet surface possesses a large number of specific, low affinity binding sites for t-PA and provide further evidence for the role of platelets in localization and regulation of fibrinolysis.     

Comparative interactions of factor IX and factor IXa with human platelets

Both factor IX and factor IXa were bound to gel filtered platelets in the presence of CaCl2 (2-20 mM) and human alpha-thrombin (0.06-0.2 units/ml) with maximal binding occurring in 10-20 min at 37 degrees C, and rapid reversibility was observed when unlabeled ligands were added in 100-fold molar excess. Competition studies with various coagulation proteins revealed that neither factor XI nor high molecular weight kininogen, at 300-fold molar excess, could compete with 125I-labeled factor IXa for binding sites on thrombin-activated platelets, whereas prothrombin and factor X, in 450-fold molar excess, could displace approximately 15 and 35%, respectively, of bound factor IXa in the absence of added factor VIII. Analysis of saturation binding data in the presence of CaCl2 and thrombin without factors VIII and X indicated the presence of 306 (+/- 57) binding sites per platelet for factor IX (Kd(app) = 2.68 +/- 0.25 nM) and 515 (+/- 39) sites per platelet for factor IXa (Kd = 2.57 +/- 0.14 nM). In the presence of thrombin-activated factor VIII (1-5 units/ml) and factor X (0.15-1.5 microM), the number of sites for factor IX was 316 (+/- 50) with Kd = 2.44 (+/- 0.30) nM and for factor IXa 551 (+/- 48) sites per platelet (Kd = 0.56 +/- 0.05 nM). Studies of competition for bound factor IXa by excess unlabeled factor IX or factor IXa, and direct 125I-labeled factor IXa binding studies in the presence of large molar excesses of factor IX, confirmed the conclusion from these studies that factor IX and factor IXa share approximately 300 low-affinity binding sites per thrombin-activated platelet in the presence of Ca2+ and in the absence of factor VIII and factor X, with an additional 200-250 sites for factor IXa with Kd(app) similar to that for factor IX. The presence of factor VIII and factor X increases by 5-fold the affinity of receptors on thrombin-activated platelets for factor IXa that participate in factor X activation.     

Divalent cations regulate glucagon binding. Evidence for actions on receptor-Ns complexes and on receptors uncoupled from Ns

The effects of Mg2+ or ethylenediaminetetraacetic acid (EDTA) on 125I-glucagon binding to rat liver plasma membranes have been characterized. In the absence of guanosine 5'-triphosphate (GTP), maximal binding of 125I-glucagon occurs in the absence of added Mg2+. Addition of EDTA or Mg2+ diminishes binding in a dose-dependent manner. In the presence of GTP, maximal binding occurs in the presence of 2.5 mM Mg2+ (EC50 = 0.3 mM) while EDTA or higher concentrations of Mg2+ diminish binding. Response to exogenous Mg2+ or EDTA depends on the concentration of Mg2+ in the membranes and may vary with the method used for membrane isolation. Solubilized 125I-glucagon-receptor complexes fractionate on gel filtration columns as high molecular weight, GTP-sensitive complexes in which receptors are coupled to regulatory proteins and lower molecular weight, GTP-insensitive complexes in which receptors are not coupled to other components of the adenylyl cyclase system. In the absence of GTP, 40 mM Mg2+ or 5 mM EDTA diminishes receptor affinity for hormone (from KD = 1.2 +/- 0.1 nM to KD = 2.6 +/- 0.3 nM) and the fraction of 125I-glucagon in high molecular weight receptor-Ns complexes without affecting site number (Bmax = 1.8 +/- 0.1 pmol/mg of protein). Thus, while GTP promotes disaggregation of receptor-Ns complexes, Mg2+ or EDTA diminishes the affinity with which these species bind hormone. In the presence of GTP, hormone binds to lower affinity (KD = 9.0 +/- 3.0 nM), low molecular weight receptors uncoupled from Ns.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pituitary somatostatin receptors. Characterization by binding with a nondegradable peptide analogue

Somatostatin receptors in the rat pituitary gland were characterized by binding analysis with a radioiodinated high affinity somatostatin analogue, 125I-Tyr1[D-Trp8]somatostatin. Receptor binding of this derivative reached equilibrium at 30 min and was maintained at a plateau for at least 60 min. Two L-Trp8- labeled somatostatin analogues. 125I-Tyr1- and [125I-Tyr11]somatostatin, displayed less stable and lower specific uptake and higher nonspecific binding. In contrast to the rapid degradation of the L-Trp8 ligands during binding assay, 125I-Tyr1]D-Trp8]somatostatin retained more than 80% of its binding activity after 90 min of incubation with pituitary particles. Pituitary particles bound 125I-Tyr1]D-Tyr8]somatostatin with high affinity (Ka = 8.6 +/- 1.2 X 10(9) M-1) and capacity of 54.4 +/- 2.6 fmol/mg. These binding sites showed specificity for the native peptide and its active analogues, and other peptide hormones, including angiotensin II, thyrotropin-releasing hormone, vasopressin, oxytocin, substance P, and gonadotropin-releasing hormone, did not inhibit tracer binding. A good correlation was observed between the binding affinities of several somatostatin analogues and their potencies as inhibitors of growth hormone release in rat pituitary cells. These findings emphasize the physiological importance of the pituitary somatostatin receptor in mediating the inhibitory action of the peptide on growth hormone release. The use of Tyr1[d-Trp8]somatostatin as a labeled ligand permits accurate determinations of the binding affinity and concentration of receptors for somatostatin in the normal pituitary gland and provides a basis for further studies of somatostatin receptor regulation and receptor-mediated cellular effects of the tetradecapeptide.     

Convulxin binding to platelet receptor GPVI: competition with collagen related peptides

Convulxin (CVX), a potent platelet aggregating protein from the venom of the snake Crotalus durissus terrificus, is known to bind to the platelet collagen receptor, glycoprotein VI (GPVI). CVX binding to human platelets was investigated by flow cytometry, using fluorescein labeled convulxin (FITC-CVX). Scatchard analysis indicated high and low affinity binding sites with Kd values of 0.6 and 4 nM and Bmax values of 1200 and 2000 binding sites per platelet. FITC-CVX binding was inhibited by collagen related peptides (CRPs) comprising a repeated GPO sequence, namely GCO(GPO)(10)GCOGNH(2) and GKO(GPO)(10)GKOGNH(2), which also bind to receptor GPVI. These peptides (monomeric or cross-linked forms) gave a high affinity inhibition of 10-20% for concentrations between 10 ng/ml and 5 microg/ml, followed by a second phase of inhibition at concentrations greater than 5 microg/ml. It was shown also that the inhibition of FITC-CVX binding by CRPs was independent on the time of preincubation of platelets with CRPs, and the same percentage of inhibition was seen with various concentrations of convulxin. Confocal microscopy of the distribution of FITC-CVX binding sites on platelets showed an homogeneous distribution of FITC-CVX bound to GPVI, although some limited clustering may exist.