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1.
pH-dependent denaturation of thrombin-activated porcine factor VIII   总被引:6,自引:0,他引:6  
Thrombin-activated porcine factor VIII (fVIIIaIIa) is a stable, active, 160-kDa heterotrimer at concentrations exceeding 2 x 10(-7) M in 0.7 M NaCl, 0.01 M histidine Cl, 5 mM CaCl2, pH 6.0, at 4 degrees C or 20 degrees C. Two of the subunits, fVIIIA1 and fVIIIA2, are derived from the heavy chain of the plasma-derived, heterodimeric fVIII precursor. The third subunit, fVIIIA3-C1-C2, is derived from the fVIII light chain. We now find that fVIIIaIIa undergoes a sharp decline in coagulant activity between pH 7 and 8. At pH 7.5, the activity of fVIIIaIIa at 3 x 10(-7) M decays within a few hours to a stable level that is approximately 70% of the value at pH 6.0, whereas at pH 8.0, greater than 99% of the activity is lost. The activity cannot be restored by readjusting the pH to 6.0. The loss of activity at pH 8.0 coincides with dissociation of the fVIIIA2 subunit since an inactive fVIIIA1/A3-C1-C2 heterodimer can be isolated by Mono S high performance liquid chromatography. After prolonged incubation at pH 8.0, the fVIIIA1 subunit also dissociates. The free fVIIIA2 fragment appears to be poorly soluble which may explain the irreversible loss of activity. Analytical velocity sedimentation of the pH-inactivated fVIIIaIIa preparation also is consistent with dissociation and precipitation of the fVIIIA2 fragment. We propose that denaturation of fVIIIaIIa by pH-dependent subunit dissociation may provide a major mechanism of inactivation of fVIIIaIIa under physiologic conditions.  相似文献   

2.
The stability of activated human and porcine factor VIII (fVIII) differ, but a direct comparison of their structural and functional properties has not been made. Highly purified, heterodimeric human recombinant and porcine plasma-derived fVIII were exchanged into a common buffer and some minor contaminants were removed by anion-exchange chromatography. The activations of human and porcine fVIII by thrombin were studied by a two-stage coagulation assay using human citrated plasma as the standard. The peak activation of porcine fVIII was 10-fold greater than human fVIII (1.1 x 10(6) unit/mg versus 1.1 x 10(5) unit/mg). The proteolytic fragmentation of fVIII by thrombin was evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was not different between human and porcine fVIII, yielding previously identified bands corresponding to fragments A1, A2, A3-C1-C2, and the B domain. Following activation by thrombin, human fVIII was subjected to cation-exchange (Mono S) high performance liquid chromatography at pH 6.0 under conditions that yields stable, heterotrimeric (A1/A2/A3-C1-C2) porcine fVIIIaIIa (Lollar, P., and Parker, C.G. (1990) Biochemistry 28, 666-674). Coagulant activity was recovered in a single peak that was less than 0.5% that of porcine fVIIIaIIa (1.2 x 10(4) unit/mg versus 2.6 x 10(6) unit/mg). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the peak fraction revealed bands corresponding to the A3-C1-C2 and A1 fragments but only trace levels of the A2 fragment. In contrast, activation of human fVIII by thrombin followed by Mono S HPLC at pH 5.0 produced a peak with 10-fold greater activity (1.2 x 10(5) unit/mg) than at pH 6.0 and which contained significant amounts of the A2 fragment. We conclude that human fVIIIIIa, like porcine fVIIIIIa, is a heterotrimer and propose that its apparent decreased coagulant activity is due to weaker association of the A2 subunit.  相似文献   

3.
Human and porcine factor VIII (fVIII) are activated by thrombin to form a heterotrimer composed of subunits designated A1 and A2 derived from the fVIII heavy chain (HC) and a subunit designated A3-C1-C2 derived from the fVIII light chain (LC). Human and porcine fVIII were activated at the same rate to the same peak levels but dissociation of the A2 subunit and concomitant loss of fVIIIa activity at pH 7.4 and 22 degrees C was 3-fold faster with human fVIIIa compared to porcine fVIIIa (0.35 min-1 versus 0.12 min-1, respectively). To determine structural requirements for the increased activity of porcine fVIII, plasma-derived hybrid human/porcine fVIII molecules were isolated. Porcine HC/human LC (pHC/hLC) fVIII had 44-fold higher coagulant activity than reconstituted human fVIII (hHC/hLC), 40-fold higher activity than hHC/pLC, and slightly (1.4-fold) higher activity than reconstituted porcine fVIII (pHC/pLC). Additionally, human and porcine A2 subunits and inactive A1/A3-C1-C2 human and porcine dimers were isolated and reconstitution experiments were done. Addition of the porcine A2 subunit to the human A1/A3-C1-C2 dimer produced coagulant activity similar to that found with porcine fVIIIa and superior to human fVIIIa. These results suggest that human fVIII has weaker coagulant activity than porcine fVIII due to faster dissociation of the A2 subunit and that the A2 subunit itself is responsible for the difference.  相似文献   

4.
Parker ET  Lollar P 《Biochemistry》2007,46(34):9737-9742
Blood coagulation factor VIII (fVIII) is activated by thrombin to form an A1/A2/A3-C1-C2 heterotrimer, which functions as a cofactor for factor IXa during intrinsic pathway factor X activation. Human thrombin-activated fVIII (fVIIIa) decays rapidly because of first-order dissociation of the A2 subunit, which may function to regulate the coagulation mechanism. The three fVIII A domains each consist of two cupredoxin-like subdomains. Substitution of the COOH-terminal A1 subdomain of porcine fVIIIa, which decays more slowly than human fVIIIa, reduces the dissociation rate constant for fVIIIa decay. Examination of a human fVIII A1-A2-A3 homology model [Pemberton, S., et al. (1997) Blood 89, 2413-2421) revealed a possible interaction between Q316 in the FG helix of the COOH-terminal A1 subdomain and M539 in the FG helix of the NH2-terminal A2 subdomain, which are sites where human and porcine fVIII differ. Decays of purified recombinant human and porcine fVIIIa and the human fVIIIa mutants Q316H, M539L and Q316H/M539L were compared at 23 and 37 degrees C. The decay rates of the Q316H and Q316H/M539L mutants, but not the M539L mutant, were significantly slower than human fVIIIa. These results indicate that the FG helix of the COOH-terminal A1 cupredoxin-like subdomain of fVIII may be under selective pressure by the requirements of hemostatic balance.  相似文献   

5.
Heterodimeric human factor VIII was proteolytically activated by catalytic levels of thrombin to yield the (labile) active cofactor factor VIIIa possessing an initial specific activity of approximately 80 units/microgram. Activation paralleled the generation of fragments A1 and A2 derived from the heavy chain and A3-C1-C2 derived from the light chain. Chromatography of factor VIIIa, on Mono-S buffered at pH 6.0 resulted in separation of the bulk of the A2 fragment from a fraction composed predominantly of A1/A3-C1-C2 dimer plus low levels of A2 fragment. Only the latter fraction contained clotting activity (approximately 20 units/microgram) which was stable and represented a less than 10% yield when compared with the peak activity of unfractionated factor VIIIa. Further depletion of A2 fragment from Mono-S-purified factor VIIIA, achieved using an immobilized monoclonal antibody to the A2 domain, yielded a relatively inactive A1/A3-C1-C2 dimer (less than 0.4 unit/microgram). Factor VIIIa (greater than 40 units/microgram) was reconstituted from the A1/A3-C1-C2 dimer plus the A2 fragment in a reaction that was Me(2+)-independent and inhibited by moderate ionic strength. Reassociation of A2 required the A1 subunit in that the A2 subunit associated weakly if at all to A3-C1-C2 in the absence of A1. These results indicated that human factor VIIIa is a trimer represented by the subunits A1/A2/A3-C1-C2 and that the A2 subunit is required for expression of factor VIIIa activity.  相似文献   

6.

Background

Human blood coagulation factor VIII (fVIII) is a large plasma glycoprotein with sequential domain arrangement in the order A1-a1-A2-a2-B-a3-A3-C1-C2. The A1, A2 and A3 domains are interconnected by long linker peptides (a1, a2 and a3) that possess the activation sites. Proteolysis of fVIII zymogen by thrombin or factor Xa results in the generation of the activated form (fVIIIa) which serves as a critical co-factor for factor IXa (fIXa) enzyme in the intrinsic coagulation pathway.

Results

In our efforts to elucidate the structural differences between fVIII and fVIIIa, we developed the solution structural models of both forms, starting from an incomplete 3.7 Å X-ray crystal structure of fVIII zymogen, using explicit solvent MD simulations. The full assembly of B-domainless single-chain fVIII was built between the A1-A2 (Ala1-Arg740) and A3-C1-C2 (Ser1669-Tyr2332) domains. The structural dynamics of fVIII and fVIIIa, simulated for over 70 ns of time scale, enabled us to evaluate the integral motions of the multi-domain assembly of the co-factor and the possible coordination pattern of the functionally important calcium and copper ion binding in the protein.

Conclusions

MD simulations predicted that the acidic linker peptide (a1) between the A1 and A2 domains is largely flexible and appears to mask the exposure of putative fIXa enzyme binding loop (Tyr555-Asp569) region in the A2 domain. The simulation of fVIIIa, generated from the zymogen structure, predicted that the linker peptide (a1) undergoes significant conformational reorganization upon activation by relocating completely to the A1-domain. The conformational transition led to the exposure of the Tyr555-Asp569 loop and the surrounding region in the A2 domain. While the proposed linker peptide conformation is predictive in nature and warrants further experimental validation, the observed conformational differences between the zymogen and activated forms may explain and support the large body of experimental data that implicated the critical importance of the cleavage of the peptide bond between the Arg372 and Ser373 residues for the full co-factor activity of fVIII.  相似文献   

7.
Association of the factor VIII light chain with von Willebrand factor   总被引:6,自引:0,他引:6  
Coagulation factor VIII (fVIII) is isolated from porcine blood as a set of three heterodimers because of proteolytic cleavages in the middle, or B region, of the parent single-chain molecule. A single 80-kDa COOH-terminal fragment, the light chain (fVIIILC), is associated with one of three forms of heavy chain (fVIIIHCs) by a calcium-dependent linkage. The purified heterodimers were dissociated using EDTA and fVIIILC, and fVIIIHCs were isolated by high pressure liquid chromatography under nondenaturing conditions. The association of fVIII, fVIIILC, and fVIIIHCs with multimeric human von Willebrand factor (vWF) was studied using analytical velocity sedimentation. A previous study using this method with an intact, single heterodimeric species of fVIII has shown that one molecule of fVIII can bind to each subunit of vWF (Lollar, P., and Parker, C.G. (1987) J. Biol. Chem. 262, 17572-17576). fVIIILC bound vWF as judged by the increase in the plateau height and sedimentation coefficient of the fVIIILC.vWF complex compared to vWF at 42,000 x g and by the decrease in the plateau height of the 4.8 S fVIIILC boundary sedimenting at 240,000 x g. Titration of a fixed concentration of fVIIILC with vWF yielded a stoichiometry of one fVIIILC molecule per subunit of vWF. Proteolytic cleavage by thrombin to remove an acidic 41-residue NH2-terminal peptide from fVIIILC completely abolished its binding to vWF. In contrast, no binding of fVIIIHCs to vWF was observed. Additionally, intact fVIII bound to vWF was completely dissociated after proteolysis by thrombin. These data are consistent with the hypothesis that a critical step in blood coagulation is the release of all regions of fVIII from vWF following a single proteolytic cleavage of fVIIILC.  相似文献   

8.
Factor VIII (fVIII) is the plasma protein that is missing or deficient in hemophilia A. In contrast, elevated levels of fVIII are associated with an increased risk of arterial and venous thrombosis. fVIII is activated by thrombin to form a non-covalently linked A1/A2/A3-C1-C2 heterotrimer. At physiological concentrations, fVIIIa decays as a result of A2 subunit dissociation, which may help regulate the balance between hemostasis and thrombosis. A2 subunit dissociation is faster in human fVIIIa than in porcine fVIIIa, which may represent an evolutionary adaptation associated with the development of the upright posture and venous stasis in the lower extremities. To investigate the basis for the different decay kinetics of human and porcine fVIIIa, hybrid fVIII molecules representing all possible combinations of human and porcine A domains were isolated. The kinetics of fVIIIa decay were measured and fit to a model describing a reversible bimolecular reaction in which the dissociation rate constant, k, and dissociation constant, Kd, were the fitted parameters. Substitution of the porcine A1 domain into human fVIIIa produced a dissociation rate constant indistinguishable from porcine fVIIIa. Subsequently, substitution of the second cupredoxin-like A1 subdomain resulted in a dissociation rate constant similar to porcine fVIIIa, whereas substitution of the first cupredoxin-like A1 subdomain resulted in a dissociation rate constant intermediate between human and porcine fVIIIa. We propose that cupredoxin-like A1 subdomains in fVIII contain inter-species differences that are a result of selective pressure on the dissociation rate constant.  相似文献   

9.
The primary structure of factor VIII consists of 2332 amino acids that exhibit 3 distinct structural domains, including a triplicated region (A domains), a unique region of 909 amino acids (B domain), and a carboxy-terminal duplicated region (C domains), that are arranged in the order A1-A2-B-A3-C1-C2. The B domain (residues 741-1648) of factor VIII is lost when factor VIII is activated by thrombin, which proteolytically processes factor VIII to active subunits of Mr 50,000 (domain A1), 43,000 (domain A2), and 73,000 (domains A3-C1-C2). To determine if the B domain is required for factor VIII coagulant activity, a variant was constructed by using recombinant DNA techniques in which residues 797-1562 were eliminated. This shortened the B domain from 909 to 142 amino acids. This variant factor VIIIdes-797-1652 was expressed in mammalian cells and was found to be functional. The factor VIIIdes-797-1562 protein was purified and shown to be processed by thrombin in the same manner as full-length factor VIII. The factor VIIIdes-797-1562 variant also bound to von Willebrand factor (vWF) immobilized on Sepharose. These results indicate that most of the highly glycosylated B domain of factor VIII is not required for the expression of factor VIII coagulant activity and its interaction with vWF.  相似文献   

10.
Development of inhibitory antibodies to coagulation factor VIII (fVIII) is the primary obstacle to the treatment of hemophilia A in the developed world. This adverse reaction occurs in 20–30% of persons with severe hemophilia A treated with fVIII-replacement products and is characterized by the development of a humoral and neutralizing immune response to fVIII. Patients with inhibitory anti-fVIII antibodies are treated with bypassing agents including recombinant factor VIIa (rfVIIa). However, some patients display poor hemostatic response to bypass therapy and improved treatment options are needed. Recently, we demonstrated that fVIII inhibitors display widely variable kinetics of inhibition that correlate with their respective target epitopes. Thus, it was hypothesized that for antibodies that display slow rates of inhibition, supplementation of rfVIIa with fVIII would result in improved thrombin generation and be predictive of clinical responses to this novel treatment regimen. In order to test this hypothesis, 10 murine monoclonal antibodies (MAbs) with non-overlapping epitopes spanning fVIII, differential inhibition titers, and inhibition kinetics were studied using a thrombin generation assay. Of the 3 MAbs with high inhibitory titers, only the one with fast and complete (classically defined as “type I”) kinetics displayed significant inhibition of thrombin generation with no improvement upon supplementation of rfVIIa with fVIII. The other two MAbs that displayed incomplete (classically defined as “type II”) inhibition did not suppress the potentiation of thrombin generation by fVIII. All antibodies that did not completely inhibit fVIII activity demonstrated potentiation of thrombin generation by the addition of fVIII as compared to rfVIIa alone. In conclusion, fVIII alone or in combination with rfVIIa corrects the thrombin generation defect produced by the majority of anti-fVIII MAbs better than single agent rfVIIa. Therefore, combined fVIII/rfVIIa therapy may provide better hemostatic control than current therapy in some patients with anti-fVIII inhibitors.  相似文献   

11.
The interaction between guanidine-activated bovine type 1 plasminogen activator inhibitor (PAI-1) and bovine vitronectin was investigated. Activated PAI-1 bound to vitronectin in a dose- and time-dependent manner, and binding was saturable. The dissociation constant (Kd) for this interaction was estimated to be 3.10(-10) mol/l by Scatchard analysis. Complexes of activated PAI-1 and vitronectin were relatively stable at 4 degrees C (T1/2 greater than 24 h), but dissociated with a T1/2 of 4 h at 37 degrees C. The half-life of PAI-1 activity was increased from 2.5 to 4.5 h upon binding to immobilized vitronectin. In order to identify the binding domain(s) in vitronectin for activated PAI-1, the ability of PAI-1 to bind to vitronectin fragments was assessed. Vitronectin was cleaved by thrombin in a dose- and time-dependent manner, generating fragments of Mr 60,000, 54,000 and 38,000. The PAI-1 binding domain(s) were not destroyed by this treatment, since the digested vitronectin competed with immobilized vitronectin for PAI-1 binding to the same extent as uncleaved vitronectin. The thrombin digested vitronectin fragments were fractionated by SDS-PAGE and analyzed by PAI-1 ligand binding. The smallest fragment (Mr 38,000) retained PAI-1 binding function, and sequence analysis demonstrated that this fragment contained the NH2-terminus of bovine vitronectin. These results suggest that the high-affinity binding site for activated PAI-1 is located in the NH2-terminal region of the bovine vitronectin molecule.  相似文献   

12.
1. Four clotting factors, Cf-1(C), Cf-2(C), Cf-1(T) and Cf-2(T) were isolated from Agkistrodon acutus (collected on mainland China and Taiwan) venom by Komori et al. (1987). It was reported that all factors possessed coagulant activity in the conversion of fibrinogen to fibrin, although they showed different chemical properties and antigenicities. 2. Their role in the clot formation system was clarified and compared with that of thrombin. Clotting factors from A. acutus venom released only fibrinopeptide A from the A alpha chain of fibrinogen, while thrombin released fibrinopeptide A and B from the A alpha and B beta chains. 3. Cf-1(C) and Cf-2(T), like thrombin, rapidly activated factor XIII in the presence of calcium ions, whereas Cf-2(C) and Cf-1(T) had little effect on factor XIII. These effects are shown by Cf-1(C) and Cf-2(T) forming a clot that remained insoluble in 8 M urea or 0.44 M monochloroacetic acid, whereas Cf-2(C) and Cf-1(T) formed a soluble clot in these agents.  相似文献   

13.
Ornithine carbamoyltransferase (OTC; subunit, 36,000 Da) [EC 2.1.3.3] is initially synthesized as a precursor (pOTC) with a transient NH2-terminal presequence of 32 amino acid residues, then is imported posttranslationally nto the mitochondrial matrix. We expressed rat pOTC in Escherichia coli, purified it in a denatured form, and showed that could be transported into isolated mitochondria in the presence of rabbit reticulocyte lysate [Murakami et al. (1988) J. Biol. Chem. 263, 18437-18442]. In order to compare the properties of the precursor and mature form of OTC, the rat mature OTC was synthesized in E. coli and purified. The recombinant OTC represented about 5% of the total bacterial protein and was present in both the supernatant and precipitate of the disrupted bacteria. The OTC, extracted from the precipitate with 8 M urea or 6 M guanidine.HCl, was essentially homogeneous, as judged by SDS-PAGE. When guanidine.HCl-denatured mature OTC was diluted and incubated at 0 degrees C for 40-60 h, it was reactivated to a specific activity of 170 mumol/min/mg protein at 37 degrees C (18% of that of the purified mature enzyme). Guanidine.HCl-denatured pOTC was activated to a specific activity of 125 mumol/min/mg protein under similar conditions. The native and reactivated OTC sedimented with an s20.w value of 6.2S, whereas the activated pOTC sedimented with an s20.w of 5.2S. The activated pOTC was more unstable than the reactivated OTC at 50 degrees C. These observations indicate that the presequence does not prevent pOTC from folding into an enzymatically active trimeric form, although the pOTC trimer appears to be less compact than the mature trimer.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

14.
Bothrops jararaca coagulation inhibitor (BjI), a protein isolated from B. jararaca plasma, specifically inhibits the coagulant activity of thrombin. Our group previously identified proteins similar to BjI in the plasma of other snakes [Tanaka-Azevedo, A.M., Tanaka, A.S., Sano-Martins I.S., 2003. A new blood coagulation inhibitor from the snake Bothrops jararaca plasma: isolation and characterization. Biochem Biophys Res Commun 308, 706-712.]. In the present study, we analyzed the presence of BjI-like proteins in the plasmas of three different species of viperid snakes, Bothrops alternatus, Bothrops jararacussu and Crotalus durissus terrificus. These proteins exhibited 109 and/or 138 kDa and were immunologically related to BjI. They also inhibited the coagulant activity of thrombin, evaluated by the thrombin time test. These findings demonstrate the presence of proteins similar to BjI in these three species, although such inhibitor could not be observed in all samples of the specimens tested. Moreover, the presence of these proteins in the plasma is related to prolongation of thrombin time, implying a relationship between these proteins and their inhibitory coagulant activity upon thrombin. Our results suggest that BjI-like proteins are widely distributed among Crotalinae snakes found in Brazil.  相似文献   

15.
The DNA polymerase of early embryos of Drosophila melanogaster has been purified to near-homogeneity. The purified enzyme gave a single, catalytically active protein band after polyacrylamide gel electrophoresis, under nondenaturing conditions. Four polypeptides with molecular weights 43,000, 46,000, 58,000, and 148,000 were resolved when this band was electrophoresed under denaturing conditions. At high ionic strengths, the DNA polymerase had a sedimentation coefficient of 8.7 S, a Stokes radius of 78 A and frictional ratio of 1.81, parameters that yield a molecular weight of 280,000. The purified DNA polymerase possessed no detectable endo- or exodeoxyribonuclease, ATPase, or RNA polymerase activity. Using an "activated" DNA template-primer, the enzyme had a pH optimum of 8.5. It was stimulated by (NH4)2SO4, KCl, and to a lesser extent, NaCl. A divalent metal cation was absolutely required; MgCl2 stimulating activity 7-fold more than MnCl2. It was inhibited by low concentrations of N-ethylmaleimide and Aphidicolon. Thus the DNA polymerase of D. melanogaster resembles most closely the alpha-DNA polymerases that have been purified from mammalian cells.  相似文献   

16.
Commercial enzymes and insect gut juice at various concentrations were used to digest Bacillus thuringiensis subsp. sotto Cry1Aa protoxin and examine the fragmentation pattern and effect on insecticidal activity. Trypsin at both high (5 mg/mL) and low (0.05 mg/mL) concentrations converted protoxin to toxin with no difference in insecticidal activity against Bombyx mori larvae. In both cases, the toxin protein had an apparent M(r) of 58.4 kDa (SDS-PAGE). Active toxin of identical M(r) was also produced with low concentrations of Pronase and subtilisin, but at high concentration, it was degraded into two protease-resistant fragments of apparent M(r) 31.8 and 29.6 kDa, and exhibited no insecticidal activity. Sequencing data established the primary cleavage site to be in domain II, the receptor-binding region of the toxin, in an exposed loop between two beta-sheet strands. Fragmentation was not observed, however, when the digests were analyzed by native protein techniques, but rather the toxin molecule appeared to be intact. The amount of activated toxin produced by Choristoneura fumiferana gut juice was markedly reduced when the gut-juice concentration was increased from 1 to 50% and correlated with a loss in insecticidal activity. However, no lower M(r) protease-resistant fragments were evident in the SDS-PAGE of these digests.  相似文献   

17.
Treatment of the catalytic (C) trimer of Escherichia coli aspartate transcarbamoylase (ATCase) with alpha-chymotrypsin by a procedure similar to that used by Chan and Enns (1978, Can. J. Biochem. 56, 654-658) has been shown to yield an intact, active, proteolytically cleaved trimer containing polypeptide fragments of 26,000 and 8,000 MW. Vmax of the proteolytically cleaved trimer (CPC) is 75% that of the wild-type C trimer, whereas Km for aspartate and Kd for the bisubstrate analog, N-(phosphonacetyl)-L-aspartate, are increased about 7- and 15-fold, respectively. CPC trimer is very stable to heat denaturation as shown by differential scanning microcalorimetry. Amino-terminal sequence analyses as well as results from electrospray ionization mass spectrometry indicate that the limited chymotryptic digestion involves the rupture of only a single peptide bond leading to the production of two fragments corresponding to residues 1-240 and 241-310. This cleavage site involving the bond between Tyr 240 and Ala 241 is in a surface loop known to be involved in intersubunit contacts between the upper and lower C trimers in ATCase when it is in the T conformation. Reconstituted holoenzyme comprising two CPC trimers and three wild-type regulatory (R) dimers was shown by enzyme assays to be devoid of the homotropic and heterotropic allosteric properties characteristic of wild-type ATCase. Moreover, sedimentation velocity experiments demonstrate that the holoenzyme reconstituted from CPC trimers is in the R conformation. These results indicate that the intact flexible loop containing Tyr 240 is essential for stabilizing the T conformation of ATCase. Following denaturation of the CPC trimer in 4.7 M urea and dilution of the solution, the separate proteolytic fragments re-associate to form active trimers in about 60% yield. How this refolding of the fragments, docking, and association to form trimers are achieved is not known.  相似文献   

18.
Activation of factor VIII by thrombin occurs via limited proteolysis at R372, R740, and R1689. The resultant active factor VIIIa molecule consists of three noncovalently associated subunits: A1-a1, A2-a2, and A3-C1-C2 (50, 45, and 73 kDa respectively). Further proteolysis of factor VIIIa at R336 and R562 by activated protein C subsequently inactivates this cofactor. We now find that the factor VIIa-tissue factor complex (VIIa-TF/PL), the trigger of blood coagulation with restricted substrate specificity, can also catalyze limited proteolysis of factor VIII. Proteolysis of factor VIII was observed at 10 sites, producing 2 major fragments (47 and 45 kDa) recognized by an anti-factor VIII A2 domain antibody. Time courses indicated the slow conversion of the large fragment to 45 kDa, followed by further degradation into at least two smaller fragments. N-Terminal sequencing along with time courses of proteolysis indicated that VIIa-TF/PL cleaved factor VIII first at R740, followed by concomitant cleavage at R336 and R372. Although cleavage of the light chain at R1689 was observed, the majority remained uncleaved after 17 h. Consistent with this, only a transient 2-fold increase in factor VIII clotting activity was observed. Thus, heavy chain cleavage of factor VIII by VIIa-TF/PL produces an inactive factor VIII cofactor no longer capable of activation by thrombin. In addition, VIIa-TF/PL was found to inactivate thrombin-activated factor VIII. We hypothesize that these proteolyses may constitute an alternative pathway to regulate coagulation under certain conditions. In addition, the ability of VIIa-TF/PL to cleave factor VIII at 10 sites greatly expands the known protein substrate sequences recognized by this enzyme-cofactor complex.  相似文献   

19.
Fibrinogen, isolated from canine plasma by the successive procedures of (1) freezing and thawing, (2) fractional precipitation with 25% saturated (HN4)2SO4 and (3) Sepharose 6B gel-filtration, had a molecular weight of 282 000 by the rapid sedimentation equilibrium method. However, a molecular weight for canine fibrinogen of 332 000, which is closer to that reported for human and bovine fibrinogens (340 000 plus or minus 20 000), was obtained from the sum of the molecular weights of the Aalpha, Bbeta and gamma chains, determined from dodecylsulfate gel electrophoretic patterns of reduced fibrinogen. Canine fibrinogen, subjected to proteolysis by urokinase-activated plasminogen for 24 h, contained degradation fragments D and E which were isolated by starch block electrophoresis and Sephadex G-200 gel-filtration. The purified D and E fragments with sedimentation coefficients of 5.0 S and 2.5 S had weight average molecular weights of 89 000 and 42 000, respectively by the rapid sedimentation equilibrium method. The ratio of D to E was 2:1 per parent fibrinogen molecule. Antigenic analysis according to anti-fibrinogen antiserum showed that both D and E fragments were antigenically deficient to native fibrinogen and revealed a reaction of non-identity with each other. Upon immunoelectrophoresis at pH 8.2, D and E had different electrophoretic mobilities. Preliminary studies indicate that based on thrombin time alone, D has anticoagulant activity while E appears to be a coagulation potentiator. Canine fibrinogen apparently consist of two core fragments with dissimilar chemical characteristics in common with the fundamental structures of human and bovine fibrinogens.  相似文献   

20.
Platelet responses to compound interactions with thrombin.   总被引:1,自引:0,他引:1  
R D Smith  W G Owen 《Biochemistry》1999,38(28):8936-8947
Catalytic and noncatalytic interactions of thrombin with platelets are investigated with use of thrombin variants with altered specificities and with ligands of thrombin receptors on platelets. Both alpha-thrombin and weakly coagulant meizothrombin-des-fragment-1 (mu-thrombin) hydrolyze proteolytically activated receptor 1 for thrombin (rPAR1(T), recombinant) with catalytic efficiencies of >10(7) M(-)(1) s(-)(1), whereas rPAR1(T) is not a substrate for weakly coagulant beta-thrombin. In contrast, both mu-thrombin and beta-thrombin are weak agonists of platelet dense body (ATP) secretion. Antibodies that block rPAR1(T) cleavage strongly inhibit the secretory reaction to alpha- and mu-thrombins but not to beta-thrombin or to thrombin receptor activating peptide (TRAP). However, catalytically inactive FPR-thrombin, which binds glycoprotein Ib but does not inhibit rPAR1(T) cleavage, inhibits responses to TRAP as well as those to alpha- and mu-thrombins, which indicates that binding of the inactive enzyme to platelets influences the function of PAR1(T). An antibody that inhibits binding of thrombin to platelet glycoprotein Ib inhibits secretory responses to thrombin but not to TRAP, so occupancy of glycoprotein Ib per se accounts for only part of the attenuation. All three thrombins stimulate a rise in cytosolic Ca(II), and the dose response to beta-thrombin is congruent with that for ATP secretion. However, the response of cytosolic Ca(II) is 10-100 times more sensitive to mu-thrombin and alpha-thrombin than ATP secretion is, and is inhibited by neither anti-PAR1(T) Ig nor FPR-thrombin. Thus, alpha-thrombin appears to have an activity not shared by either mu- or beta-thrombins. This activity is owed to more than coupling of independent signals from cleavage of two proteolytically activated receptors, as there is no synergism when mu-thrombin and beta-thrombin costimulate secretion. It is concluded either that alpha-thrombin has a third interaction site on platelets with which neither mu-thrombin nor beta-thrombin interacts or that dual receptors are coordinately cleaved. In either case, the strong secretory response to thrombin appears to be moderated, independently of cytosolic Ca(II), by occupancy of a noncatalytic interaction site such as glycoprotein Ib.  相似文献   

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