Purification and properties of a prothrombin activator from the venom of Notechis scutatus scutatus

The prothrombin activator present in the venom of the mainland tiger snake (Notechis scutatus scutatus) was purified to homogeneity by gel chromatography on Sephadex G-200 followed by ion-exchange chromatography on SP-Sephadex. The venom activator has an apparent molecular weight of 54,000. It consists of a heavy chain (Mr = 32,000) and a light chain (Mr = 23,000) held together by one or more disulfide bridges. The active site is located at the heavy chain region of the molecule. The venom activator contains 8 gamma-carboxyglutamic acid residues/molecule. Gel electrophoretic analysis of prothrombin activation indicates that the venom activator is capable of cleaving both the Arg 274-Thr 275 and Arg 323-Ile 324 bonds of bovine prothrombin. The order of bond cleavage appears to be random since prethrombin-2 and meizothrombin occur as intermediates during prothrombin activation. Prothrombin activation by the venom activator alone is very slow. This is explained by the unfavorable kinetic parameters for the reaction (Km for prothrombin = 105 microM, Vmax = 0.0025 nmol of prothrombin activated per min/microgram of venom activator). Phospholipids plus Ca2+ and Factor Va greatly stimulate venom-catalyzed prothrombin activation. In the presence of 50 microM phospholipid vesicles composed of 20 mol % phosphatidylserine and 80 mol % phosphatidylcholine, the Km drops to 0.2 microM, whereas there is hardly any effect on the Vmax. Factor Va causes a 3,500-fold increase of the Vmax (8.35 nmol of prothrombin activated per min/microgram of venom activator) and a 10-fold decrease of the Km (9.5 microM). The most favorable kinetic parameters are observed in the presence of both 50 microM phospholipid and Factor Va (Km = 0.16 microM, Vmax = 27.9 nmol of prothrombin activated per min/microgram of venom activator). These changes of the kinetic parameters explain the stimulatory effects of Factor Va and phospholipid on venom-catalyzed prothrombin activation. The venom activator slowly converts the Factor Xa-specific chromogenic substrates CH3SO2-D-leucyl-glycyl-L-arginine-p-nitroanilide and N-benzoyl-L-isoleucyl-L-glutamyl-(piperidyl)-glycyl-L-arginyl-p-nitroani lide hydrochloride. Factor Va causes a 7-fold stimulation of chromogenic substrate conversion by the venom activator. This stimulation appears to be the result of the formation of a tight 1:1 complex between the venom activator and Factor Va.     

Structural and functional characterization of a prothrombin activator from the venom of Bothrops neuwiedi

A prothrombin activator from the venom of Bothrops neuwiedi was purified by gel filtration on Sephadex G-100, ion-exchange chromatography on DEAE-Sephacel and affinity chromatography on a Zn2+-chelate column. The overall purification was about 200-fold, which indicates that the prothrombin activator comprises about 0.5% of the crude venom. The venom activator is a single-chain protein with an apparent molecular weight of 60 kDa. It readily activated bovine prothrombin with a Km of 38 microM and a Vmax of 120 mumol prothrombin activated per min per mg of venom activator. Venom-catalyzed prothrombin activation was not accelerated by the so-called accessory components of the prothrombinase complex, phospholipids plus Ca2+ and Factor Va. Gel-electrophoretic analysis of prothrombin activation indicated that the venom activator only cleaved the Arg-323-Ile-324 bond of bovine prothrombin, since meizothrombin was the only product of prothrombin activation. The activator did not hydrolyze commercially available p-nitroanilide substrates and its prothrombin-converting activity was not inhibited by benzamidine, phenylmethylsulfonyl fluoride, dansyl-Glu-Gly-Arg-chloromethyl ketone and soy-bean trypsin inhibitor. However, chelating agents such as EDTA, EGTA and o-phenanthroline rapidly destroyed the enzymatic activity of the venom activator. The activity of chelator-treated venom activator could be partially restored by the addition of an excess CaCl2. These results indicate that the venom activator remarkably differs from Factor Xa and that the enzyme is not a serine proteinase, but likely belongs to the metalloproteinases. The structural and functional properties of the venom prothrombin activator from B. neuwiedi are similar to those reported for the venom activator from Echis carinatus.     

Purification and characterization of a prothrombin activator from the venom of the Australian brown snake, Pseudonaja textilis textilis

A simple procedure, involving chromatography on concanavalin A-Sepharose and gel filtration, has been developed for the purification of a prothrombin activator from the venom of the Australian brown snake Pseudonaja textilis textilis. The prothrombin activator, which is a major venom component, is a high molecular weight protein (Mr greater than or equal to 200,000) which yields a number of subunits when examined by SDS-PAGE. It is related antigenically to the venom prothrombin activator of the taipan Oxyuranus scutellatus. P. textilis prothrombin activator is able to coagulate citrated plasma, warfarin plasma, and Factor V- and Factor X-deficient plasmas; to convert purified human prothrombin to thrombin; and to hydrolyse the peptide p-nitroanilide substrate S-2222. Calcium ions and phospholipids had little if any effect on the rates of coagulation of citrated plasma or S-2222 hydrolysis catalysed by this enzyme.     

Kinetic studies of prothrombin activation: effect of factor Va and phospholipids on the formation of the enzyme-substrate complex

The kinetic parameters of bovine prothrombin activation by factor Xa were determined in the absence and presence of factor Va as a function of the phospholipid concentration and composition. In the absence of factor Va, the Km for prothrombin increases proportionally with the phospholipid concentration and correlates well with the affinity of prothrombin for the different membranes. Phospholipid vesicles with a high affinity for prothrombin yield low Km values compared to membranes with less favorable binding parameters. At limited phospholipid concentrations, the Vmax of prothrombin activation correlates with the binding affinity of factor Xa for the various phospholipid vesicles. Membranes with a high affinity for factor Xa have high Vmax values, while for membranes with a low affinity a low Vmax is observed. Extrapolation of double-reciprocal plots of 1/Vmax vs. 1/[phospholipid] to infinite phospholipid concentrations, a condition at which all factor Xa would participate in prothrombin activation, yields a kcat of 2-4 min-1 independent of the type and amount of acidic phospholipid present in the vesicles. Also, in the presence of factor Va the Km for prothrombin varies proportionally with the phospholipid concentration. There is, however, no correlation between the binding parameters and the Km. Factor Va drastically lowers the Km for prothrombin for vesicles that have a low affinity for prothrombin. Vesicles composed of 20 mol % phosphatidylglycerol and 80 mol % phosphatidylcholine have a Km of 0.04 microM when factor Va is present, compared to 2.2 microM determined in the absence of factor Va.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of a Ca2+-calmodulin-stimulated cyclic GMP phosphodiesterase from bovine brain

A calmodulin-stimulated form of cyclic nucleotide phosphodiesterase from bovine brain has been extensively purified (1000-fold). Its specific activity is approximately 4 mumol min-1 (mg of protein)-1 when 1 microM cGMP is used as the substrate. This form of calmodulin-sensitive phosphodiesterase activity differs from those purified previously by showing a very low maximum hydrolytic rate for cAMP vs. cGMP. The purification procedure utilizing ammonium sulfate precipitation, ion-exchange chromatography on DEAE-cellulose, gel filtration on Sephacryl S-300, isoelectric focusing, and affinity chromatography on calmodulin-Sepharose and Cibacron blue-agarose results in a protein with greater than 80% purity with 1% yield. Kinetics of cGMP and cAMP hydrolysis are linear with Km values of 5 and 15 microM, respectively. Addition of calcium and calmodulin reduces the apparent Km for cGMP to 2-3 microM and increases the Vmax by 10-fold. cAMP hydrolysis shows a similar increase in Vmax with an apparent doubling of Km. Both substrates show competitive inhibition with Ki's close to their relative Km values. Highly purified preparations of the enzyme contain a major protein band of Mr 74 000 that best correlates with enzyme activity. Proteins of Mr 59 000 and Mr 46 000 contaminate some preparations to varying degrees. An apparent molecular weight of 150 000 by gel filtration suggests that the enzyme exists as a dimer of Mr 74 000 subunits. Phosphorylation of the enzyme preparation by cAMP-dependent protein kinase did not alter the kinetic or calmodulin binding properties of the enzyme. Western immunoblot analysis indicated no cross-reactivity between the bovine brain calmodulin-stimulated gGMP phosphodiesterase and the Mr 60 000 high-affinity cAMP phosphodiesterase present in most mammalian tissues.     

Purification of a protein C activator from the venom of the southern copperhead snake (Agkistrodon contortrix contortrix)

A protease has been purified by ion-exchange chromatography from the venom of Agkistrodon contortrix contortrix (Southern copperhead snake) that can activate the vitamin K dependent protein, protein C. The apparent molecular weight of this protease, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was 20,000 under nonreducing conditions. Incubation of this protease with plasma resulted in a prolongation of the clotting time and a time-dependent increase in amidolytic activity. Incubation of the protease with purified protein C resulted in an increase in both amidolytic and anticoagulant activity. The protease had no inhibitory effect on thrombin, factor V, fibrinogen, or factor X. It had slight clotting activity toward fibrinogen. The apparent Km of the protease for protein C was 0.28 microM. Calcium ions were observed to inhibit protein C activation with an apparent Ki of 0.2 mM. Ethylenediaminetetraacetic acid, diisopropyl fluorophosphate, and soybean trypsin inhibitor were observed to inhibit the venom protease. These results suggest that the venom of the Southern copperhead snake contains a protease that is a specific activator of protein C.     

Purification and properties of an activating enzyme of blood clotting factor X from the venom of Cerastes cerastes

An activator of blood coagulation factor X was found in the venom of the horned viper Cerastes cerastes, and was purified by gel filtration, ion-exchange chromatography and chromatofocussing. The activator is a protein composed of a heavy and a light polypeptide chain linked by disulfide bonds. Two subforms of the activator were found. Both contained a heavy chain of Mr 58000 and are distinguished from each other by the presence of two different light chains of Mr 17700 and 15000. The activator appears to cleave the bond in the factor X molecule that is also cleaved by factor IXa. Factor X activation by the activator is strongly stimulated by Ca2+. The kinetic parameters for the activation reaction have been determined. A Km for factor X of 19.2 nM and a Vmax of 0.11 pmol of Xa/min per ng venom were found.     

Purification and characterization of oxalyl-coenzyme A decarboxylase from Oxalobacter formigenes.

Oxalyl-coenzyme A (oxalyl-CoA) decarboxylase was purified from Oxalobacter formigenes by high-pressure liquid chromatography with hydrophobic interaction chromatography, DEAE anion-exchange chromatography, and gel permeation chromatography. The enzyme is made up of four identical subunits (Mr, 65,000) to give the active enzyme (Mr, 260,000). The enzyme catalyzed the thiamine PPi-dependent decarboxylation of oxalyl-CoA to formate and carbon dioxide. Apparent Km and Vmax values, respectively, were 0.24 mM and 0.25 mumol/min for oxalyl-CoA and 1.1 pM and 0.14 mumol/min for thiamine pyrophosphate. The maximum specific activity was 13.5 microM oxalyl-CoA decarboxylated per min per mg of protein.     

Purification and characterization of 17 beta-hydroxysteroid dehydrogenase from Cylindrocarpon radicicola

An NAD+-linked 17 beta-hydroxysteroid dehydrogenase was purified to homogeneity from a fungus, Cylindrocarpon radicicola ATCC 11011 by ion exchange, gel filtration, and hydrophobic chromatographies. The purified preparation of the dehydrogenase showed an apparent molecular weight of 58,600 by gel filtration and polyacrylamide gel electrophoresis. SDS-gel electrophoresis gave Mr = 26,000 for the identical subunits of the protein. The amino-terminal residue of the enzyme protein was determined to be glycine. The enzyme catalyzed the oxidation of 17 beta-hydroxysteroids to the ketosteroids with the reduction of NAD+, which was a specific hydrogen acceptor, and also catalyzed the reduction of 17-ketosteroids with the consumption of NADH. The optimum pH of the dehydrogenase reaction was 10 and that of the reductase reaction was 7.0. The enzyme had a high specific activity for the oxidation of testosterone (Vmax = 85 mumol/min/mg; Km for the steroid = 9.5 microM; Km for NAD+ = 198 microM at pH 10.0) and for the reduction of androstenedione (Vmax = 1.8 mumol/min/mg; Km for the steroid = 24 microM; Km for NADH = 6.8 microM at pH 7.0). In the purified enzyme preparation, no activity of 3 alpha-hydroxysteroid dehydrogenase, 3 beta-hydroxysteroid dehydrogenase, delta 5-3-ketosteroid-4,5-isomerase, or steroid ring A-delta-dehydrogenase was detected. Among several steroids tested, only 17 beta-hydroxysteroids such as testosterone, estradiol-17 beta, and 11 beta-hydroxytestosterone, were oxidized, indicating that the enzyme has a high specificity for the substrate steroid. The stereospecificity of hydrogen transfer by the enzyme in dehydrogenation was examined with [17 alpha-3H]testosterone.     

Studies of the role of factor Va in the factor Xa-catalyzed activation of prothrombin, fragment 1.2-prethrombin-2, and dansyl-L-glutamyl-glycyl-L-arginine-meizothrombin in the absence of phospholipid

In order to specifically evaluate the role of Factor Va in the prothrombinase complex, studies of the activation of prothrombin, Fragment 1.2-prethrombin-2, and active-site-blocked meizothrombin were carried out, both in the absence of phospholipid and at concentrations of substrates and Factor Va sufficient to approach saturation in all components. Km values were independent of Factor Va concentrations, whereas kcat (apparent) values approached saturation with respect to Factor Va concentrations. The three respective substrates exhibited the following parameters of kinetics (Km, microM; kcat, s-1 at saturating [Factor Va]): prothrombin (9.0 +/- 0.4; 31 +/- 1); Fragment 1.2-prethrombin-2 (5.4 +/- 0.4; 13 +/- 2); and meizothrombin (3.6 +/- 0.3; 51 +/- 5). Models of kinetics were constructed to interpret the results, and two of these were formally consistent with experimental results. Both models indicated that the variation of kcat(app) with concentrations of Factor Va reflects the formation of a Factor Va-Factor Xa binary complex. Analysis of kinetics indicated Kd values for this interaction of 1.3 +/- 0.1, 3.0 +/- 0.5, and 1.0 +/- 0.1 microM for the three respective substrates. The models differed in the interpretation of Km. One indicated that Km reflects a binary interaction between Factor Xa and prothrombin, whereas the other indicated a binary interaction between Factor Va and prothrombin. Both indicated that two of the three possible binary interactions between the three components would be reflected in Km and kcat values but not the third. To distinguish these models, the binary interactions were studied by extrinsic fluorescence (Va.Xa), light-scattering (Factor Va.prothrombin), and competition kinetics (Xa.II). The first two interactions were detected and were characterized by Kd values of 2.7 +/- 0.1 microM (Va.Xa) and 8.8 +/- 0.8 microM (Factor Va.prothrombin). No active-site-dependent interaction between prothrombin and Factor Xa could be detected in the absence of Factor Va. The results of these studies suggest that Factor Va interacts with both Factor Xa and prothrombin and effectively presents one to the other in the formation of a ternary enzyme-substrate-cofactor complex. In addition, a comparison of the parameters of kinetics of conversion of prothrombin and its intermediates indicates that meizothrombin is the major intermediate of prothrombin activation in the absence, as well as in the presence of phospholipid.     

The interaction of bone Gla protein (osteocalcin) with phospholipid vesicles

The binding interaction of bone Gla protein (BGP), or osteocalcin, to phospholipid vesicles in the presence of calcium has been investigated. Two separate indirect methodologies involving displacement of pyrene-modified Factor Va bound to phospholipid vesicles, and competition with several coagulation proteins in a prothrombin activation assay were performed. Titration of BGP into a cuvette containing phospholipid vesicles (75:25, L-alpha-phosphatidylcholine/L-alpha-phosphatidylserine (PCPS] saturated with pyrene-modified Factor Va resulted in a systematic decrease in steady-state anisotropy, suggesting competition for membrane binding sites with pyrene-modified Factor Va. BGP was also found to inhibit thrombin generation in the prothrombin activation assay. Approximately 50% inhibition was observed at 3 microM BGP under phospholipid-limiting (0.5 microM PCPS) concentrations. No inhibition was observed under phospholipid excess (30 microM PCPS) concentrations. Direct measurement of phospholipid binding was measured using equilibrium gel filtration. Elution profiles using fixed lipid (3.4 mumol of PCPS) and varying BGP concentrations (1-17 microM) in the presence of 3 mM CaCl2 showed a BGP-phospholipid association. Quantitation of determined isotherm yielded a dissociation constant of 6 +/- 1 microM with a stoichiometry of 102 +/- 9 BGP molecules/vesicle at saturation (35 PCPS lipids/BGP) in the presence of 3 mM CaCl2. These results support the hypothesis that protein gamma-carboxylation events are coincident with membrane binding potential.     

Proteolysis of factor Va by factor Xa and activated protein C

Bovine Factor Va, produced by selective proteolytic cleavage of Factor V by thrombin, consists of a heavy chain (D chain) of Mr = 94,000 and a light chain (E chain) of Mr = 74,000. These peptides are noncovalently associated in the presence of divalent metal ion(s). Each chain is susceptible to proteolysis by activated protein C and by Factor Xa. Sodium dodecyl sulfate electrophoretic analysis indicates that cleavage of the E chain by either activated protein C or Factor Xa yields two major fragments: Mr = 30,000 and Mr = 48,000. Amino acid sequence analysis indicates that the Mr = 30,000 fragments have identical NH2-terminal sequences and that this sequence corresponds to that of intact E chain. The Mr = 48,000 fragments also have identical NH2-terminal sequences, indicating that activated protein C and Factor Xa cleave the E chain at the same position. Sodium dodecyl sulfate electrophoretic analysis indicates that activated protein C cleavage of the D chain yields two products: Mr = 70,000 and Mr = 24,000. Amino acid sequence analysis indicates that the Mr = 70,000 fragment has the same NH2-terminal sequence as intact D chain, whereas the Mr = 24,000 fragment does not. Factor Xa cleavage of the D chain also yields two products: Mr = 56,000 and Mr = 45,000. The Mr = 56,000 fragment corresponds to the NH2-terminal end of the D chain and Factor V. Functional studies have shown that both chains of Factor Va may be entirely cleaved to products by Factor Xa without loss of activity, whereas activated protein C cleavage results in loss of activity. Since activated protein C and Factor Xa cleave the E chain at the same position, the cleavage of the D chain by activated protein C is responsible for the inactivation of Factor Va.     

Effect of membrane fluidity and fatty acid composition on the prothrombin-converting activity of phospholipid vesicles.

Vesicles composed of phospholipids with different fatty acyl side chains have been utilized to examine the importance of the nonpolar membrane region for the prothrombin-converting activity of procoagulant phospholipid vesicles. Membranes composed of phosphatidylserine (PS) and phosphatidylcholine (PC) with unsaturated fatty acyl side chains were more active in prothrombin activation than membranes composed of phospholipids with saturated fatty acyl chains. This phenomenon was observed above the phase transition temperature, i.e., on membranes in the liquid-crystalline state. The prothrombin-converting activity of saturated phospholipids approached the activity of unsaturated phospholipids at high factor Va concentrations, which is indicative for a less favorable equilibrium constant for prothrombinase assembly on membrane surfaces composed of saturated phospholipids. The difference between saturated and unsaturated phospholipids was annulled on membranes with high mole percentages of PS. This may result from a compensating contribution of electrostatic forces to the binding equilibria involved in prothrombinase assembly. Additional effects on the prothrombin-converting activity were observed when membranes containing saturated phospholipids were studied below their phase transition temperature. In agreement with Higgins et al. [(1985) J. Biol. Chem. 260, 3604-3612], we found that the time required for the assembly of prothrombinase from membrane-bound factors Xa and Va is considerably prolonged on solid membranes. However, we also observed an effect of membrane fluidity on the steady-state rate of prothrombin activation. Kinetic experiments at saturating factor Va concentrations showed that the transition from the liquid-crystalline to the gel state caused a more than 9-fold decrease of the kcat of prothrombin activation without affecting the Km for prothrombin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enzymes of vitamin B6 degradation. Purification and properties of 4- and 5-pyridoxolactonases

4-Pyridoxolactone and 5-pyridoxolactone, formed by dehydrogenation of pyridoxal or isopyridoxal during the bacterial degradation of vitamin B6 by Pseudomonas MA-1 and Arthrobacter Cr-7, respectively, are hydrolyzed to the corresponding acids by distinct inducible lactonases which were purified to homogeneity. 4-Pyridoxolactonase from Pseudomonas MA-1 has an Mr of 54,000 and contains two probably identical subunits of Mr = 28,600. It has a pH optimum of 7.0, a Km of 5.9 microM, and a Vmax at 25 degrees C of 35.2 mumol X min-1 X mg-1. 5-Pyridoxolactonase from Arthrobacter Cr-7 has an Mr of 65,200 and also contains two probably identical subunits of Mr = 32,800. It has a pH optimum of 7.1-7.7, a Km of 300 microM, and a Vmax at 25 degrees C of 21.5 mumol-1 X min-1 X mg-1. The two lactonases require no added cofactors or metal ions; their activities are inhibited by sulfhydryl reagents but are not affected by metal-chelating reagents. Although the two lactonases are entirely specific for their respective substrates, 4-pyridoxolactone is a competitive inhibitor (KI = 52 microM) for 5-pyridoxolactonase, and 5-pyridoxolactone is a competitive inhibitor (KI = 48 microM) for 4-pyridoxolactonase.     

Protein S is essential for the activated protein C-catalyzed inactivation of platelet-associated factor Va

The prothrombin-converting activity of Factor Xa was enhanced by thrombin-stimulated Factor V-deficient platelets and supplementary extraneous Factor Va, and also by thrombin-stimulated normal human platelets. Both extraneous Factor Va and intra-platelet Factor Va were equally inactivated by a gamma-carboxyglutamic acid-containing plasma protease, activated protein C. However, a relatively larger amount of activated protein C was required for efficient inactivation of platelet-associated Factor Va as compared with the amount of activated protein C needed for inactivation of phospholipid vesicle-associated Factor Va. Protein S, another gamma-carboxyglutamic acid-containing plasma protein, increased the rate of the inactivation of platelet-associated Factor Va about 25-fold. This stimulating effect was observed only slightly with the thrombin-modified protein S. Thus, it was concluded that protein S is essential for the process of inactivation of platelet-associated Factor Va by activated protein C.     

Characterization of a protein C activator from Agkistrodon contortrix contortrix venom

The protease from Southern Copperhead venom that activates protein C was purified to homogeneity by sulfopropyl (SP)-Sephadex C-50 ion-exchange chromatography, Sephadex G-150 gel filtration, and Mono-S fast protein liquid chromatography. The purified enzyme is a glycoprotein containing 16% carbohydrate, and migrated as a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular mass of 40,000 kDa. The enzyme is composed of a single polypeptide chain possessing an NH2-terminal sequence of Val-Ile-Gly-Gly-Asp-Glu-Cys-Asn-Ile-Asn-Glu-His. The purified venom protein C activator hydrolyzed several tripeptide p-nitroanilides. The amidolytic and proteolytic activities of the enzyme were readily inhibited by phenylmethanesulfonyl fluoride, p-amidinophenylmethanesulfonyl fluoride, chloromethyl ketones, and human antithrombin III. Covalent binding of diisopropyl fluorophosphate to the enzyme was confirmed using a tritium-labeled preparation of the inhibitor. The venom protease readily activated human and bovine protein C at 1:1000 enzyme:substrate weight ratio. The protease also cleaved human prothrombin, factor X, factor IX, factor VII, and fibrinogen. Prothrombin coagulant activity decreased upon incubation with the venom protease, and the rate of this reaction was reduced in the presence of calcium. Factor X and factor IX coagulant activity increased upon incubation with the venom protease in the presence of calcium, and decreased in the absence of calcium. Human factor VII clotting activity decreased slightly upon incubation with the venom protease. Although the venom protease did not clot human fibrinogen, it nonetheless cleaved the A alpha chain of fibrinogen, and this cleavage appeared to be associated with a measurable increase in the clottability of the protease-treated fibrinogen by thrombin. These data demonstrate that the protein C activator from Southern Copperhead venom is a typical serine protease with a relatively broad specificity.     

Purification and characterization of a novel form of 20 alpha-hydroxysteroid dehydrogenase from Clostridium scindens.

We have purified a steroid-inducible 20 alpha-hydroxysteroid dehydrogenase from Clostridium scindens to apparent homogeneity. The final enzyme preparation was purified 252-fold, with a recovery of 14%. Denaturing and nondenaturing polyacrylamide gradient gel electrophoresis showed that the native enzyme (Mr, 162,000) was a tetramer composed of subunits with a molecular weight of 40,000. The isoelectric point was approximately pH 6.1. The purified enzyme was highly specific for adrenocorticosteroid substrates possessing 17 alpha, 21-dihydroxy groups. The purified enzyme had high specific activity for the reduction of cortisone (Vmax, 280 nmol/min per mg of protein; Km, 22 microM) but was less reactive with cortisol (Vmax, 120 nmol/min per mg of protein; Km, 32 microM) at pH 6.3. The apparent Km for NADH was 8.1 microM with cortisone (50 microM) as the cosubstrate. Substrate inhibition was observed with concentrations of NADH greater than 0.1 mM. The purified enzyme also catalyzed the oxidation of 20 alpha-dihydrocortisol (Vmax, 200 nmol/min per mg of protein; Km, 41 microM) at pH 7.9. The apparent Km for NAD+ was 526 microM. The initial reaction velocities with NADPH were less than 50% of those with NADH. The amino-terminal sequence was determined to be Ala-Val-Lys-Val-Ala-Ile-Asn-Gly-Phe-Gly-Arg. These results indicate that this enzyme is a novel form of 20 alpha-hydroxysteroid dehydrogenase.     

Construction, expression, and characterization of a chimera of factor IX and factor X. The role of the second epidermal growth factor domain and serine protease domain in factor Va binding.

The prothrombinase complex, which catalyzes the conversion of prothrombin to thrombin, consists of activated Factor X, Factor Va, a membrane surface and Ca2+. To examine the structures that support Factor Va binding to Factor X, we used in vitro mutagenesis to construct a chimeric molecule that includes regions of Factor IX and Factor X. This chimera (IXGla,E1XE2,SP) was prepared from cDNA encoding the second epidermal growth factor (EGF) and serine protease domains of Factor X linked downstream from the cDNA encoding the signal peptide, propeptide, Gla domain, and first EGF domain of Factor IX. The cDNAs encoding the Factor IX/X chimera and wild-type Factor X were each expressed in Chinese hamster ovary cells and the secreted proteins purified by affinity chromatography using polyclonal anti-Factor X antibodies. The chimera migrated as a single major band corresponding to a molecular weight of 68,000. By Western blotting, the chimeric protein stained with both polyclonal anti-Factor X and anti-Factor IX antibodies. gamma-Carboxyglutamic acid analysis demonstrated near complete carboxylation of both the wild-type Factor X and the Factor IX/X chimera. Compared with Factor X, the rate of zymogen activation of the Factor IX/X chimera was about 50% that of Factor X when activated by Factor IXa, Factor VIIIa, phospholipid, and Ca2+. The enzyme form of the Factor IX/X chimera, activated Factor IX/X, generated using the coagulant protein of Russell's viper venom, expressed full amidolytic activity compared with Factor Xa. The activated Factor IX/X chimera had about 14% of the activity of Factor Xa when employed in a prothrombinase assay; this activity reached 100% with increasing concentrations of Factor Va. A binding assay was employed to test the ability of the active site-inactivated Factor IX/Xa chimera to inhibit the binding of Factor Xa to the Factor Va-phospholipid complex, thus inhibiting the activation of prothrombin to thrombin. In this assay the active site-inactivated form of the chimera competed with Factor Xa completely but with decreased affinity for the Factor Va-phospholipid complex. These data indicate that the second EGF domain and the serine protease domain of Factor Xa are sufficient to interact with Factor Va. The Factor IX/X chimera is a good substrate for the tenase complex; the defective enzymatic activity of the activated Factor IX/X chimera can be accounted for by its decreased affinity for Factor Va relative to Factor Xa.     

Inactivation of factor Va by plasmin

The inactivation of Factor Va by plasmin was studied in the presence and absence of phospholipid vesicles and calcium ions. The cleavage patterns of bovine Factor Va and its isolated subunits were analyzed using polyacrylamide gel electrophoresis, and the progress of inactivation was monitored by clotting assays and measurements of prothrombin activation using 5-dimethylaminonaphthalene-1-sulfonylarginine-N-(3-ethyl-1,5-penta nediyl)amide. In addition, the ability of prothrombin and Factor Xa to protect Factor Va from inactivation by human plasmin was examined. The data presented indicate that the cofactor Factor Va is inactivated rapidly upon its interaction with human plasmin. The rate of inactivation is significantly enhanced in the presence of phospholipid vesicles, suggesting that the inactivation process is a membrane-bound phenomenon. The isolated D component (heavy chain of factor Va) was found to be slowly degraded by human plasmin, giving rise to cleavage products different from those obtained with activated protein C and Factor Xa. However, the 48- and 30-kDa fragments obtained from human plasmin degradation of component E (light chain of Factor Va) appear to be similar to those obtained following the proteolysis of the same subunit by activated protein C and Factor Xa.     

Proexosite-1-dependent recognition and activation of prothrombin by taipan venom

An activator complex from the venom of Oxyuranus scutellatus scutellatus (taipan venom) is known to rapidly activate prothrombin to thrombin. To determine whether, similar to prothrombinase, taipan venom utilizes proexosite-1 on prothrombin for a productive complex assembly, the activation of proexosite-1 mutants of prethrombin-1 by the partially purified venom was studied. It was discovered that basic residues of this site (Arg(35), Lys(36), Arg(67), Lys(70), Arg(73), Arg(75), and Arg(77)) are also crucial for recognition and rapid activation of the substrate by taipan venom. This was evidenced by the observation that the K(m) and k(cat) values for the activation of the charge reversal mutants of prethrombin-1 (in particular K36E, R67E, and K70E) were markedly impaired. Competitive kinetic studies with the Tyr(63)-sulfated hirudin(54-65) peptide revealed that although the peptide inhibits the activation of the wild type zymogen by taipan venom with a K(D) of approximately 2 microm, it is ineffective in inhibiting the activation of mutant zymogens (K(D) > 4-30 microm). Interestingly, an approximately 50-kDa activator, isolated from the taipan venom complex, catalyzed the activation of prothrombin in a factor Va-dependent manner and exhibited identical activation kinetics toward the substrate in the presence of the hirudin peptide. These results suggest that, similar to prothrombinase, proexosite-1 is a cofactor-dependent recognition site for taipan venom.