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PubMed1990

Interaction between lipids and bovine brain calmodulin: lysophosphatidylcholine-induced conformation change.

We have monitored the interaction of several lipids with the bovine brain calmodulin(CaM) and analyzed the effect of lysophosphatidylcholine(lyso-PC, 2-50 micrograms/ml) on conformation of CaM and the interaction between CaM and CaM-binding protein(CaMBP), using a fluorescence signal of 1-(dimethylamino)naphthalene-5-sulfonate-labeled CaM(DNS-CaM). Lyso-PC(egg, 20 micrograms/ml), among various natural lipids including phosphatidylserine(PS), phosphatidylinositol(PI), phosphatidylethanolamine (PE) and their lyso forms, greatly and dose-dependently enhanced the intensity of DNS fluorescence of DNS-CaM in the presence (100 microM CaCl2) and absence (1 mM EGTA) of Ca2+. Apparent dissociation constants calculated from the fluorometric titrations of binding of lyso-PC to DNS-CaM were 0.6 and 3.7 micrograms/ml in the presence and absence of Ca2+, respectively. Lyso-PC remarkably prevented both trypsin-induced quenching of the fluorescence of DNS-CaM and tryptic digestion of native CaM in the absence of Ca2+. Enhancement of DNS fluorescence of DNS-CaM by CaMBP was observed only in the presence of Ca2+ and lyso-PC could further increase the fluorescence intensity of the complex. These all results suggest that lyso-PC can modulate the interaction between CaM and CaMBP as a result of its direct effect on conformation of CaM.

Animals↗
PubMed1987

Presence of proteins and glutamate as major constituents of the venom of the spider Araneus gemma.

The venom from the spider Araneus gemma contains an inhibitor of physiologic glutamate receptors and of glutamate binding sites in brain synaptic membranes. In the present study the chemical composition of the venom was examined in order to determine the presence of constituents that may have physiologically important actions on the prey. The milked venom contains high concentrations of protein (approximately equal to 200 micrograms/microliter) and of glutamate (130-425 mM) and very low concentrations of epinephrine, epinine, dopamine and 3,4-dihydroxyphenylacetic acid. There is also marked heterogeneity in the venom peptides detected by SDS gel electrophoresis. The presence of free glutamate may be very important for the actions of the glutamate receptor inhibitor in the venom.

Amino Acids↗
PubMed1982

Cleavage and activation of human prothrombin by Echis carinatus venom.

The cleavage of human prothrombin by partially purified Echis carinatus venom (ECV) was investigated in the present report. Incubation of prothrombin with ECV resulted in the rapid cleavage of prothrombin to alpha-thrombin, with the release of fragment-1 and fragment-2. When dansyl arginine-N-(3-ethyl-1,5-pentanediyl) amide (DAPA), a very effective inhibitor of thrombin, was included in the ECV-prothrombin solution, meizothrombin was rapidly formed. Only small amounts of meizothrombin-1 could be detected. Prolonged incubation (23 h) in the presence of DAPA, however, resulted in nearly quantitative conversion of meizothrombin to meizothrombin-1 and fragment-1. Kinetic studies strongly suggested that the conversion of meizothrombin to meizothrombin-1 was due to ECV and not meizothrombin autolysis. In addition, EDTA, which inhibits ECV, blocked the cleavage of meizothrombin. Amino terminal sequence analysis indicated that ECV cleaves human prothrombin at two sites; Gly158-Ser159 and Arg322-Ile323. The former site differs from the site of autolytic cleavage of meizothrombin which occurs at Arg155-Ser156. In contrast to reports in the literature, the results of the present study indicate that the release of fragment-1 does not precede activation of human prothrombin by ECV.

Amino Acid Sequence↗
PubMed1988

Influence of divalent and monovalent cations on some active site properties of human factor Xa.

The effect of divalent and monovalent cations on the hydrolysis of BzIleGlu(OR)GlyArgpNA(S-2222) was compared to the rate of inactivation of factor Xa by dansyl-GluGlyArg-chloromethylketone(DERG-CK). At substrate concentrations below Km, an approximate four-fold increase in amidase activity was observed in the presence of manganese ions while a three-fold increase was observed with calcium ions. The presence of magnesium ions resulted in a two-fold increase in amidase activity. Similar increases in the rate of inactivation of factor Xa by DERG-CK were observed. Na+ ions had a marked enhancing effect of both factor Xa amidase activity and inactivation by DERG-CK. Kinetic parameters for the hydrolysis of S-2222 by factor Xa were obtained in the presence and absence of Ca++ and Na+. Vmax values increased in the presence of either Ca++ or Na+. Km values increased in the presence of Ca++ while there was a modest decrease in Km in the presence of Na+. It is suggested that the enhanced activity of factor Xa is a reflection of changes in the reactivity of active site residues.

Amino Acid Chloromethyl Ketones↗
PubMed1988

Effect of thrombin inhibitors on thrombin-induced platelet release and aggregation.

Thrombin-induced platelet activation was interrupted with hirudin or Dansylarginine N-(3-ethyl-1-5-pentanediyl) amide (DAPA) to study the time requirement for receptor occupancy by thrombin in promoting platelet responses at low (0.25 U/ml), intermediate (0.5 U/ml) and high (1 U/ml) thrombin concentrations. Each of these thrombin inhibitors suppressed adenosine triphosphate (ATP) release and aggregation by thrombin when added either before or simultaneously with thrombin or within seconds of the initiation of these responses by thrombin. If the inhibitors were added later, yet before aggregation or release was complete, no effect was present. The period of time for which active thrombin was required in order to promote these reactions had the following characteristics: (i) it is thrombin concentration dependent for a given response; (ii) it is longer for aggregation than for ATP secretion at each thrombin concentration; (iii) it is increased in platelets modified by chymotrypsin or platelets partially inhibited by antimycin A and 2-deoxy-D-glucose, which have prolonged aggregation and ATP release responses. In direct comparison studies, the inhibitory effects of hirudin and DAPA were identical on aggregation and ATP release. Thrombin binding, under similar experimental conditions identical to those used to measure platelet activation, was prevented by hirudin, but not by DAPA. Therefore, the effect of DAPA on thrombin must be at the proteolytic site region and not at the hirudin-inhibitable platelet binding region. It is concluded from these studies that the tight coupling requirements for thrombin to induce platelet dense granule release and aggregation are directly dependent upon both the thrombin concentration and the rate of the individual platelet responses. Catalytic site integrity is required for the duration of this period of receptor occupancy.

Adenosine Triphosphate↗
PubMed1989

The interaction of thrombin with platelet protease nexin.

Thrombin interacts with a platelet protein which is immunologically related to fibroblast protease nexin and has been termed platelet protease nexin I (PNI). Conflicting hypotheses about the relationship of the thrombin-PNI complex formation to platelet activation have been proposed. The studies presented here demonstrate that the platelet-associated and supernatant complexes with added 125I-thrombin are formed only under conditions which produce platelet activation in normal and chymotrypsin-modified platelets. The platelet-associated complex is formed prior to the appearance of complexes in supernatants. Appearance of the supernatant complex coincides with the appearance of thrombospondin in the reaction supernatants. Excess native thrombin, dansylarginine N-(3-ethyl-1,5-pentanediyl) amide or hirudin can prevent radiolabeled platelet-associated complex formation if added before 125I-thrombin. DAPA or hirudin can prevent or dissociate complex formation if added up to one minute after thrombin but not at later time points. The surface associated complex is accessible to trypsin although a portion remains with the cytoskeletal proteins when thrombin-activated platelets are solubilized with Triton X 100. The surface-associated complex formation parallels many aspects of the specific measurable thrombin binding, yet it does not appear to involve other identified surface glycoprotein thrombin receptors or substrates. Although the time course of appearance of the complexes in supernatants is consistent with other data which suggest that PNI may be released from platelet granules during platelet activation, other explanations for the appearance of PNI on the platelet surface and in supernatants during platelet activation are possible.

Amyloid beta-Protein Precursor↗
PubMed1995

Endotoxin-induced pulmonary vascular injury is mainly mediated by activated neutrophils in rats.

Acute respiratory failure is a common complication in patients with disseminated intravascular coagulation associated with sepsis. To elucidate the role of coagulation abnormalities in acute lung injury in sepsis, we investigated the effect of anticoagulants on the pulmonary vascular injury in rat induced by lipopolysaccharide (LPS). When administered intravenously, LPS (5 mg/kg body weight) significantly increased the accumulation of 111indium-labeled neutrophils in lung 30 min after administration. Subsequently, the pulmonary vascular permeability and the serum level of fibrin and fibrinogen degradation products (E) [FDP (E)] increased and remained elevated for several hours. Neither heparin alone, heparin plus antithrombin III, or dansyl-Glu-Gly-Arg-chloromethyl ketone-treated factor Xa, a selective inhibitor of thrombin generation, prevented LPS-induced vascular injury 6 hours after LPS administration, whereas these substances significantly inhibited the increase in serum FDP (E) at that time. LPS-induced pulmonary vascular injury was significantly attenuated in rats with methotrexate-induced leukocytopenia or treated with ONO-5046, a potent granulocyte elastase inhibitor, although ONO-5046 did not inhibit the LPS-induced increase in serum FDP (E). Thus, activated leukocytes play a more important role than coagulation abnormalities in the pathogenesis of LPS-induced pulmonary vascular injury in an experimental rat model of endotoxemia.

Amino Acid Chloromethyl Ketones↗