Biomedical subjects

I Schvartz

Publications and source records attributed to I Schvartz.

At least 19 recordsLinked to original sources

PubMed2001

Localization of protein kinase A and vitronectin in resting platelets and their translocation onto fibrin fibers during clot formation.

Physiological stimulation of platelets with thrombin brings about the release of protein kinase A (PKA) into the plasma. In human blood, this kinase singles out and phosphorylates vitronectin (Vn), a multifunctional regulatory protein, which was proposed to play an important role in the control of fibrinolysis. Here we present immuno-cytochemical evidence to show: (i) that intact platelets possess on their surface an ecto-PKA which can preferentially phosphorylate Vn; (ii) that in the resting platelet, both the catalytic and the regulatory subunits of PKA are present on the platelet surface, in the surface-connected canalicular system, and within the alpha-granules of the platelets; (iii) that the process initiated upon platelet activation, which leads to the formation of fibrin fibers and consequently forms the fibrin net, is accompanied by a translocation of PKA, of Vn, and of PAI-1 onto the fibrin fibers. We propose that the localization and the translocation of these proteins in the fibrin net, together with our finding that PKA phosphorylation of Vn reduces its grip of PAI-1, can unleash PAI-1 in its free form. The free PAI-1 can then assume its latent (non inhibitory) conformation, allow plasminogen activators to trigger the formation of active plasmin, and to initiate fibrinolysis.

Biological Transport↗

PubMed2000

Snapping of the carboxyl terminal tail of the catalytic subunit of PKA onto its core: characterization of the sites by mutagenesis.

A set of 45 mutants of the carboxyl terminal tail of the PKA catalytic subunit was prepared and used to assess the contribution of this tail to the structure and function of the kinase. Ala substitutions of Asp 323, Phe 327, Glu 333, and Phe 350 resulted in a complete loss of enzymatic activity. Other replacements by Ala (Phe 314, Tyr 330, Glu 332, and Phe 347) brought about either a drop in activity to less than 10% of the wild-type enzyme or a reduction of affinity toward ATP (Lys 317, Lys 319, Tyr 330, and Glu 332) or toward Kemptide (Ile 315, Tyr 330, Val 337, Ile 339, Lys 345, and Glu 346). Mutations of Ser 338, a major autophosphorylation site of PKA, by Ala, Glu, Asp, Gln, and Asn showed that the kinetic parameters of these mutants are similar to those of the wild-type. The contribution of each of these tail mutations to the structure and stability of the kinase was assessed by monitoring its effect on the heat stability (when measurable) or by determining the susceptibility of the mutant kinase to cleavage by the Kinase Splitting Membranal Proteinase/Meprin beta. Here we show that the tail of PKA has a key role in creating the active conformation of the kinase. It does so by means of specific amino acid residues, which act as "snapping points" to embrace the two lobes of the kinase and orient them in the correct juxtaposition for substrate docking, biorecognition, and catalysis.

Adenosine Triphosphate↗

PubMed1999

Vitronectin.

Vitronectin is a multifunctional glycoprotein present in blood and in the extracellular matrix. It binds glycosaminoglycans, collagen, plasminogen and the urokinase-receptor, and also stabilizes the inhibitory conformation of plasminogen activation inhibitor-1. By its localization in the extracellular matrix and its binding to plasminogen activation inhibitor-1, vitronectin can potentially regulate the proteolytic degradation of this matrix. In addition, vitronectin binds to complement, to heparin and to thrombin-antithrombin III complexes, implicating its participation in the immune response and in the regulation of clot formation. The biological functions of vitronectin can be modulated by proteolytic enzymes, and by exo- and ecto-protein kinases present in blood. Vitronectin contains an RGD sequence, through which it binds to the integrin receptor alpha v beta 3, and is involved in the cell attachment, spreading and migration. Antibodies against alpha v beta 3 or synthetic peptides containing an RGD sequence are now being tested as therapeutic agents in the treatment of human cancers, bone diseases (e.g. osteoporosis) and in pathological disorders which involve angiogenesis.

Animals↗

PubMed1995

Vitronectin overrides a negative effect of TNF-alpha on astrocyte migration.

Morphogenesis and tissue repair require appropriate cross-talk between the cells and their surrounding milieu, which includes extracellular components and soluble factors, e.g., cytokines and growth factors. The present work deals with this communication needed for recovery after axotomy in the central nervous system (CNS). The failure of CNS axons to regenerate after axonal injury has been attributed, in part, to astrocyte failure to repopulate the injury site. The goal of this work was to provide an in vitro model to mimic the in vivo response of astrocytes to nerve injury and to find ways to modulate this response and create a milieu that favors astrocyte migration and repopulation of the injury site. In an astrocyte scratch wound model, we blocked astrocyte migration by tumor necrosis factor alpha (TNF-alpha). This effect could not be reversed by astrocyte migration-inducing factors such as transforming growth factor beta 1 (TGF-beta 1) or by any of the tested extracellular matrix (ECM) components (laminin and fibronectin) except for vitronectin (Vn). Vn, added together with TNF-alpha, counteracted the TNF-alpha blockage and allowed a massive migration of astrocytes (not due to cell proliferation) beyond that allowed by Vn only. Heparan sulfate proteoglycans (HSPG) were shown to be involved in the migration. The results may be relevant to regeneration of CNS axons, and may also provide an example that an extracellular component (Vn) can overcome and neutralize a negative effect of a growth factor/cytokine (TNF-alpha) and can act in synergy with other features of this cytokine to promote a necessary function (e.g., cell migration) that is otherwise inhibited.

Animals↗

PubMed1993

Evidence for an extra-cellular function for protein kinase A.

In addition to its intra-cellular functions, cAMP-dependent protein kinase (PKA) may well have an extra-cellular regulatory role in blood. This suggestion is based on the following experimental findings: (a) Physiological stimulation of blood platelets brings about a specific release of PKA, together with its co-substrates ATP and Mg++; (b) In human serum, an endogenous phosphorylation of one protein (p75, M(r) 75 kDa) occurs; this phosphorylation is enhanced by addition of cAMP and blocked by the Walsh-Krebs specific PKA inhibitor; (c) No endogenous phosphorylation of p75 occurs in human plasma devoid of platelets, but the selective labeling of p75 can be reproduced by adding to plasma the pure catalytic subunit of PKA; (d) p75 was shown to be vitronectin (V), a multifunctional protein implicated in processes associated with platelet activation, and thus a protein whose function may require modulation for control; (e) The phosphorylation of vitronectin occurs at one site (Ser378) which, at physiological pH, is buried in its two-chain form (V65 + 10) but it becomes 'exposed' in the presence of glycosaminoglycans (GAGs) e.g. heparin or heparan sulfate. Such a transconformation may be used for targeting the PKA phosphorylation to vitronectin molecules bound to GAGs, for example in the extracellular matrix or on cell surfaces; (f) From the biochemical point of view (Km values and physiological concentrations) the phosphorylation of vitronectin can take place at the locus of a hemostatic event; (g) The phosphorylation of Ser378 in vitronectin alters its function, since it significantly reduces its ability to bind the inhibitor-1 of plasminogen activator(s) (PAI-1).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1992

PDGF induces tyrosine phosphorylation in osteoblast-like cells: relevance to mitogenesis.

We have studied the association between protein tyrosine phosphorylation and the mitogenic effect induced by platelet-derived growth factor (PDGF) in the osteoblast-like cell line MC3T3-E1. PDGF caused a dose-dependent increase in [3H]thymidine incorporation in MC3T3-E1 cells, reaching a plateau at 10 ng/ml. Vanadate, a potent phosphatase inhibitor, induced a twofold increase in thymidine incorporation. The combination of vanadate and PDGF resulted in a dose-dependent synergistic effect on thymidine incorporation. Genistein, a tyrosine kinase inhibitor, inhibited in a dose-related manner (2-20 microM) the mitogenic effect induced by either PDGF or the combination of vanadate and PDGF. These observations suggest that tyrosine kinases are involved in mediating the mitogenic effect of PDGF in these cells. PDGF treatment of MC3T3-E1 cells and subsequent immunoblotting with antiphosphotyrosine antibodies resulted in a marked phosphorylation of the PDGF receptor. Vanadate had a lesser effect on PDGF receptor phosphorylation, but given together with PDGF it induced a significant increase in the intensity of receptor phosphorylation. Preincubation with genistein abrogated these effects. Taken together, these findings indicate a direct correlation between thymidine incorporation and tyrosine phosphorylation in MC3T3-E1 cells and suggest that tyrosine phosphorylation plays a role in PDGF-induced mitogenic activity in osteoblast-like cells.

Animals↗

PubMed1991

Direct evidence for multiple endothelin receptors.

Competition binding experiments and peptide mapping techniques were employed in order to directly address the possible existence of endothelin (ET) receptor subtypes in the atria. Competition binding assays for 125I-labeled ET-1 or 125I-labeled ET-3 to bovine atrial membrane preparations suggest the existence of two ET receptor subtypes, one of which binds ET-1 and ET-3 with a similar affinity while the other shows preference for ET-3. However, cross-linking experiments of both peptides to this tissue resulted in the identification of a single 50-kDa protein. To identify directly the existence of multiple ET receptors, peptide mapping of cross-linked 125I-labeled ET-1 or 125I-labeled ET-3 receptors was conducted. Different peptide maps were obtained only under conditions that preferentially label one receptor subtype. These results indicate, for the first time, the existence of two ET receptor subtypes in the atria which differ from each other in both their binding characteristics and primary structure.

Animals↗

PubMed1991

Identification of a single binding protein for endothelin-1 and endothelin-3 in bovine cerebellum membranes.

Competition binding experiments of [125I]endothelin-3 ([125I]ET-3) to bovine cerebellum membrane preparations in the presence of either ET-3 or ET-1 have indicated the presence of a single class of high affinity binding sites for these two peptides in the brain. Cross-linking of [125I]ET-3 to cerebellum membrane preparations with dissuccinimidyl suberate (DSS) resulted in the labeling of two bands with apparent mol wt of 52 and 30 kDa. Under these conditions the labeling intensities of these two bands were similar. However, addition of 5 mM EDTA to the protease inhibitor mixture during membrane preparations as well as the binding and cross-linking reaction increased the labeling of the 52-kDa protein while reducing the labeling of the 30-kDa protein. Peptide map comparisons of the 52- and 30-kDa protein bands using Staphylococcus aureus V8 protease and papain revealed that the 30-kDa band is a proteolytic degradation product of the 52-kDa protein. These results suggest that the 52-kDa protein represents the specific binding protein of ET-3, and thus, the apparent mol wt of the ET receptor is 50 kDa, subtracting the mol wt of the iodinated ET. Since cross-linking of [125I]ET-1 to cerebellum membrane preparations revealed the same two bands of 52 and 30 kDa, peptide mapping of the 52-kDa proteins, cross-linked with either [125I]ET-1 or ET-3, was conducted. Under these experimental conditions, identical peptide fragments were generated by both Staphylococcus aureus V8 protease and papain. These results suggest that ET-1 and ET-3 bind to a common brain binding protein with an apparent mol wt of 50 kDa.

Animals↗

PubMed1990

Identification of endothelin receptors by chemical cross-linking.

Specific binding sites for human/porcine endothelin have been found in rat brain membrane preparations using an [125I]endothelin. The apparent Kd value was 3 +/- 2 nM with a Bmax value of 2,200 +/- 200 fmol/mg protein. Chemical cross-linking of [125I]endothelin to rat brain membranes by using the cross-linking reagent disuccinimidyl suberate (DSS) revealed two specific proteins of Mr = 52,000 and Mr = 30,000. The same results were obtained by photoaffinity labeling of [125I]azidoendothelin to rat brain membranes. The labeling of the two proteins was inhibited in a dose-dependent manner by unlabeled endothelin but not by unrelated peptides. Peptide map comparisons of the Mr = 52,000 and Mr = 30,000 protein bands using Staphylococcus aureus V8 protease and papain revealed that the Mr = 30,000 band is a proteolytic degradation product of the Mr = 52,000. The apparent mol wt of the endothelin receptor is, therefore, 50,000, subtracting the mol wt of the iodinated endothelin.

Affinity Labels↗

PubMed1990

Bovine cerebellum endothelin receptor: solubilization and identification.

Endothelin receptors were solubilized from bovine cerebellum membrane preparations in an active form by using the zwitterionic detergent CHAPS, [3-(3-cholamidopropyl)dimethylammonio]-1-propane sulfonic acid. The solubilized receptors displayed high affinity, saturability, and specificity. The dissociation constant (Kd) for endothelin was 7 +/- 2 nM, and the number of binding sites was 600 +/- 200 fmol/mg protein. These results are similar to those obtained for the membrane-bound receptor and suggest that during solubilization the binding characteristics of the receptor are preserved. Attempts to purify the solubilized receptors in an active form using affinity chromatography techniques, i.e. Affi-gel 15 column coupled to endothelin, were not successful. Nevertheless, identification of the receptors was achieved by affinity chromatography of the solubilized proteins and subsequent iodination. Autoradiographic analysis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a major protein with an apparent mol wt of 50 kD. Taken together with our previous findings, this result suggests that the 50-kD band represents the endothelin receptor.

Animals↗

PubMed1989

Effect of guanine nucleotides on phospholipase C activity in permeabilized pituitary cells: possible involvement of an inhibitory GTP-binding protein.

Cultured pituitary cells prelabeled with myo-[2-3H] inositol were permeabilized by ATP4-, exposed to guanine nucleotides and resealed by Mg2+. Addition of guanosine 5'-0-(3-thio triphosphate) (GTP gamma S) to permeabilized cells, or gonadotropin releasing hormone (GnRH) to resealed cells, resulted in enhanced phospholipase C activity as determined by [3H] inositol phosphate (Ins-P) production. The effect was not additive, but the combined effect was partially inhibited by guanosine 5'-0-(2-thiodiphosphate) (GDP beta S) or by neomycin. Surprisingly, addition of GDP beta S (100-600 microM) on its own resulted in a dose-related increase in [3H]Ins-P accumulation. Several nucleoside triphosphates stimulated phospholipase C activity in permeabilized pituitary cells with the following order: UTP greater than GTP gamma S greater than ATP greater than CTP. The stimulatory effect of UTP, ATP and CTP, but not GTP gamma S or GDP beta S, could also be demonstrated in normal pituitary cells suggesting a receptor-activated mechanism. GTP and GTP gamma S decreased the affinity of GnRH binding to pituitary membranes and stimulated LH secretion in permeabilized cells. These results suggest the existence of at least two G-proteins (stimulatory and inhibitory) which are involved in phospholipase C activation and GnRH action in pituitary cells.

Animals↗

PubMed1989

Competitive interaction between endothelin and sarafotoxin: Binding and phosphoinositides hydrolysis in rat atria and brain.

Binding studies with the structurally similar vasoconstrictor peptides 125I-endothelin and 125I-sarafotoxin b, the former of mammalian origin and the latter derived from snake venom, reveal their mutually exclusive binding to rat atrium and various regions of the rat brain. In these tissues endothelin, like sarafotoxin, induces phosphoinositide hydrolysis which is in part Ca2+-independent. It is suggested that endothelins and sarafotoxins share common binding sites and mechanisms of action.

Animals↗

PubMed1987

Internalization and recycling of receptor-bound gonadotropin-releasing hormone agonist in pituitary gonadotropes.

The fate of cell surface gonadotropin-releasing hormone (GnRH) receptors on pituitary cells was studied utilizing lysosomotropic agents and monensin. Labeling of pituitary cells with a photoreactive GnRH derivative, [azidobenzoyl-D-Lys6]GnRH, revealed a specific band of Mr = 60,000. When photoaffinity-labeled cells were exposed to trypsin immediately after completion of the binding, the radioactivity incorporated into the Mr = 60,000 band decreased, with a concomitant appearance of a proteolytic fragment (Mr = 45,000). This fragment reflects cell surface receptors. Following GnRH binding, the hormone-receptor complexes underwent internalization, partial degradation, and recycling. The process of hormone-receptor complex degradation was substantially prevented by lysosomotropic agents, such as chloroquine and methylamine, or the proton ionophore, monensin. Chloroquine and monensin, however, did not affect receptor recycling, since the tryptic fragment of Mr = 45,000 was evident after treatment with these agents. This suggests that recycling of GnRH receptors in gonadotropes occurs whether or not the internal environment is acidic. Based on these findings, we propose a model describing the intracellular pathway of GnRH receptors.

Affinity Labels↗

PubMed1987

Effect of phorbol ester on stimulus-secretion coupling mechanisms in gonadotropin releasing hormone-stimulated pituitary gonadotrophs.

The feedback regulatory control mechanism exerted by activated Ca2+/phospholipid-dependent protein C kinase upon gonadotropin releasing hormone (GnRH) binding, stimulation of phosphoinositide turnover and gonadotropin secretion was investigated in cultured pituitary cells. Addition of the tumor promoter phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), at concentrations which activate pituitary protein C kinase, to cultured pituitary cells resulted in up-regulation of GnRH receptors (155% at 4 h). The stimulatory effect of GnRH on [3H]inositol phosphates (Ins-P) production in myo-[2-3H]inositol prelabeled pituitary cells was not inhibited by prior treatment of the cells with TPA (10(-9)-10(-7) M). Higher concentrations of TPA (10(-6)-10(-5) M) inhibited the effect of GnRH on [3H]Ins-P production. Increasing concentrations of TPA or the permeable analog of diacylglycerol 1-oleoyl-2-acetylglycerol (OAG) stimulated luteinizing hormone (LH) release from cultured pituitary cells with ED50 values of 5 x 10(-9) M and 10 micrograms/ml, respectively. No consistent inhibition or additivity of LH release was observed when increasing doses of TPA or OAG were added with a submaximal dose of GnRH. These results suggest that protein C kinase might mediate the known homologous up-regulation of GnRH receptors during the reproductive cycle. Protein C kinase is positively involved in mediating the process of gonadotropin secretion. Unlike many other systems, activation of protein C kinase in pituitary gonadotrophs is not involved in negative feed-back regulation of stimulus-secretion-coupling mechanisms in GnRH-stimulated gonadotrophs.

Animals↗

PubMed1987

Production and characterization of antibodies to gonadotropin-releasing hormone receptor.

Antibodies to the gonadotropin-releasing hormone (GnRH) receptor of bovine pituitary membranes have been raised in rabbits by immunization with affinity-purified receptor preparations. These antibodies did not compete with 125I-labeled GnRH analog (Buserelin) for binding to the receptors but did precipitate rat and bovine solubilized receptors labeled with 125I-Buserelin. Binding of the antibodies to the receptors was also demonstrated by immunoprecipitation of 125I-labeled purified receptors and photoaffinity-labeled receptors. The antibodies did not have a GnRH-like activity but rather inhibited, in a dose-dependent manner, GnRH-stimulated luteinizing hormone release from cultured rat pituitary cells. In addition, the antibodies did not inhibit luteinizing hormone release stimulated by high K+ concentration. This suggests that the antibodies recognize domains of the receptor other than the binding site of the hormone and thereby inhibit the biological response. These GnRH receptor antibodies provide a useful tool for studying GnRH receptor structure, function, localization, and biosynthesis.

Animals↗

PubMed1986

Wheat germ agglutinin behaves as a GnRH antagonist but induces gonadotrope desensitization.

Preincubation of cultured rat pituitary cells with 10 micrograms/ml of either wheat germ agglutinin (WGA) or concanavalin A inhibited LH release stimulated with GnRH (0.5 nM) by 55% and 40%, respectively. WGA-inhibition of LH release stimulated by GnRH was dose-dependent, reaching a plateau of 75% inhibition at 50 micrograms/ml. Concomitantly, WGA induced a dose-dependent inhibition of 125I-Buserelin specific binding to pituitary cells, with a maximal inhibition of 45%. The inhibition of 125I-Buserelin binding by WGA is due to GnRH receptor internalization and not to persistent occupancy of the receptors. In addition to the effect of WGA on receptor internalization, WGA also induced partial desensitization of pituitary cells but was ineffective in modulating GnRH-induced desensitization. These findings indicate that WGA has all the characteristics of a GnRH antagonist, nevertheless, it does induce desensitization of cultured rat pituitary cells to further stimulation with GnRH.

Animals↗

PubMed1986

Solubilization and purification of rat pituitary gonadotropin-releasing hormone receptor.

Gonadotropin-releasing hormone (GnRH) receptors were solubilized from rat pituitary membrane preparations in an active form by using the zwitterionic detergent CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid). The solubilized receptor exhibits high affinity, saturability, and specificity. The soluble supernatant retained 100% of the original binding activity when stored at 4 or -20 degrees C in the presence of 10% glycerol. The receptors were resolved into two components on the basis of chromatography on wheat germ agglutinin-agarose. Homogeneous receptor preparation was obtained by two cycles of affinity chromatography on immobilized avidin column coupled to [biotinyl-D-Lys6]GnRH. The overall recovery of the purified receptor was 4-10% of the initial activity in the CHAPS extract, and the calculated purification -fold was approximately 10,000 to 15,000. Analysis of iodinated purified GnRH receptors by autoradiography indicated the presence of two bands, Mr = 59,000 and 57,000. This was confirmed by photoaffinity labeling of the partially purified receptors and suggests that both components can specifically bind the hormone.

Animals↗

PubMed1986

Copper induces luteinizing hormone release and desensitization of pituitary gonadotropes.

Copper stimulated LH release from cultured rat pituitary cells in a dose-and time-dependent manner. After 4 h of incubation with 10 mu M Cu2+, LH release was stimulated by 3-fold. The release of LH stimulated by Cu2+ was Ca2+ dependent, thus excluding the possibility that the releasing activity of this divalent cation was due to a toxic effect on pituitary cells. The stimulatory action of Cu2+ is substantially mediated via the GnRH-receptors since Cu2+ inhibited 125I-Buserelin binding and since GnRH-antagonist blocked most of the Cu2+-stimulated LH release (80%). Both GnRH (1 microM) and Cu2+ (10 microM) induced desensitization of pituitary cells to a subsequent stimulation of either GnRH (0.5 nM) or Cu2+ (10 microM). However, in contrast to GnRH, Cu2+ did not induce down regulation of GnRH receptors. These findings suggest that the Cu2+ effects are mainly mediated through the GnRH receptors.

Animals↗