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51.
Catalytic competence of human alpha- and gamma-thrombin in the activation of fibrinogen and factor XIII 总被引:4,自引:0,他引:4
Steady-state kinetic parameters were compared for the action of alpha- and gamma-thrombin on the physiologically important thrombin substrates fibrinogen and factor XIII at 37 degrees C, pH 7.4, and 0.14 M NaCl. gamma-Thrombin, an alpha-thrombin derivative proteolytically cleaved at R-B73 and K-B154, was observed to catalyze the release of fibrinopeptide A (FPA) from fibrinogen with a specificity constant (kcat/Km) of 5 X 10(3) M-1 s-1. This value was approximately 2400-fold lower than the specificity constant for the corresponding alpha-thrombin-catalyzed reaction. The low specificity constant was attributed to an increase in Km and a decrease in kcat for gamma-thrombin-catalyzed release of FPA from fibrinogen. Conversion of alpha-thrombin to gamma-thrombin also resulted in an approximately 800-fold reduction in the specificity constant for thrombin-catalyzed release of fibrinopeptide B (FPB) from fibrin I, as well as a loss in discriminatory power. Whereas alpha-thrombin preferentially released FPA from intact fibrinogen, gamma-thrombin released FPA and FPB from intact fibrinogen at similar rates. In contrast to the large difference in specificity constants observed for alpha- and gamma-thrombin catalysis with fibrin(ogen) as substrate, the specificity constant (2.6 X 10(4) M-1 s-1) observed for gamma-thrombin-catalyzed release of activation peptide from factor XIII was only 5-fold lower than the corresponding value for the alpha-thrombin-catalyzed reaction. Additionally, the promotion of factor XIII activation by fibrin characteristic of the alpha-thrombin-catalyzed reaction did not occur in the gamma-thrombin-catalyzed reaction.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
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Summary The uptake of deoxyguanosine by rat liver mitochondria was characterized. The process required an intact mitochondrial membrane and exhibited a dependence on added phosphate. Deoxyguanosine uptake was minimally influenced by Mg2+ or Mn2+, but Ca2+ at concentrations above 0.5 mM were detrimental. Of the deoxynucleosides tested, only deoxyinosine inhibited the uptake of deoxyguanosine. The ribonucleoside guanosine was not observed to compete with its deoxynucleoside analog. Known inhibitors of nucleoside transport, cytochalasin B and NBMPR, did not block deoxyguanosine uptake, but the sulfhydryl reagents NEM and pCMB were both inhibitory. The uptake of deoxyguanosine was shown to be a saturable process and an apparent Km of 0.64 M was calculated from a Hanes plot. 相似文献
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The mechanism of antigen receptor gene assembly 总被引:25,自引:0,他引:25
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Five cladoceran zooplankton species in a Venezuelan floodplainlake, Laguna la Orsinera, were analyzed to identify factorscontrolling their dynamics over a period encompassing isolation,inundation by the Orinoco River and drainage. The abundanceof each species increased rapidly to a maximum associated withflooding of the lake by the Orinoco River. We calculated birth,death and population growth rates, and developed a method forestimating uncertainty in these parameters, in order to interpretobserved cladoceran abundance patterns. Rapid increases in populationsizes resulted both from high birth rates associated with decreasingturbidity following inundation and from the hatching of restingeggs. Birth rates remained high throughout the study, but highdeath rates restricted each population to a brief maximum andthen maintained small population sizes throughout the remainderof the study. Our demographic analyses suggest that high mortalityresulted from intense predation by fish and by Chaoborus larvae,rather than from resource limitation, and that this mortalitywas the major factor regulating cladoceran abundance duringinundation and drainage in this tropical floodplain lake. 相似文献
59.
Incorporation of acetate and arachidonic acid into lipid classes was examined in chorioallantoic membranes obtained from sheep at Day 24 of pregnancy. Conceptus tissues were incubated in vitro with 5 mM acetate, 0.042 mM arachidonate, 0.45 muCi [1-14C]acetate, and 5.0 muCi [5,6,8,9,11,12,14,15-3H]arachidonate for 3 and 6 h. After incubation, tissue lipid fractions were extracted, isolated, and examined for radiolabel incorporations. Medium was extracted and analyzed for radiolabeled metabolites. Metabolic pathways commonly associated with fatty acid metabolism were confirmed to be present. Acetate was utilized for de novo synthesis of free cholesterol and free fatty acid. Fatty acids containing radiolabel from both acetate and arachidonate were mainly esterified in phospholipid and triglyceride, major lipid classes found in chorioallantoic tissue. Labeled metabolites of acetate were not sufficient for analytical measurement in medium. Metabolites of arachidonic acid from lipoxygenase and cyclooxygenase pathways were determined in medium after incubation. Results suggest that, within Day 24 ovine chorioallantoic tissue, utilization of exogenous arachidonate and de novo lipogenesis from acetate function in a parallel and anabolic mode appropriate for membrane expansion. 相似文献
60.
David F. V. Lewis Costas Ioannides Dennis V. Parke 《Chemico-biological interactions》1989,70(3-4):263-280
Molecular dimensions and molecular orbital calculations of the electronic structures of 56 substrates, inhibitors and inducers of the cytochromes P-448 and other families of the cytochromes P-450 are reported. Substrates of the cytochromes P-448 are shown to be planar molecules with relatively large values of area/depth2, and to have electronic structures with relatively low values for ΔE, the difference in energy between the frontier orbitals (E(LEMO) − E(HOMO)). Substrates of other families of the cytochromes P-450 are globular molecules, with relatively low values of area/depth2 and relatively high values of ΔE. Molecular orbital calculations of the active oxygen species, singlet oxygen and superoxy anion, have also been made. Singlet oxygen is a poor electron donor (low values of E(HOMO)) but a good electron acceptor (low values of E(LEMO)), whereas superoxy anion is a good electron donor and a poor electron acceptor. Cytochrome P-448 substrates, which are good electron donors, would preferentially accept singlet oxygen, a good electron acceptor; substrates of the other families of cytochrome P-450, which are less effective electron donors, would preferentially accept superoxy anion, a good electron donor, although substrates of both cytochromes P-448 and other P-450s may accept both species of active oxygen. Together with recent published evidence, these data provide a greater understanding of the mode of activation of oxygen by the various families of the cytochromes P-450, and to the insertion of active oxygen into the substrates. Mechanisms are proposed for the oxygenation of substrates, namely, epoxidation involving singlet oxygen and hydroxylation by superoxy anion. Finally, a detailed explanation of the cytochrome P-450 cycle is discussed, and mechanisms of the different types of oxidative metabolism are presented. 相似文献