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.     

The activation of factor V by factor Xa or alpha-chymotrypsin and comparison with thrombin and RVV-V action. An improved factor V isolation procedure.

Bovine plasma factor V has been isolated by a preparative procedure involving barium sulfate adsorption, QAEC extraction, poly(ethylene glycol) precipitation, and finally chromatography on a desulfated Sepharose 6B column. Factor V was recovered as a single peak in yields of 35-40% with a specific activity of 50-70 representing a purification of 1000-2000-fold relative to the starting plasma. The apparent molecular weight of the purified factor V was 439,000 +/- 5000. On sodium dodecyl sulfate gel and analytical gel electrophoresis, this factor V preparation showed multiple bands, but results are inconclusive with regard to a possible subunit structure for this factor. The purified factor V was stable for at least 1-2 weeks when stored at 4 degrees C in 0.2 M Tris-acetate, 50 mM CaCl2, 10% glycerol, pH 7.5. When stored at -20 degrees C in 50% glycerol, this preparation was stable for several months. Treatment of the purified factor V with bovine factor Xa, RVV-V, thrombin, or chymotrypsin (but not trypsin) led to a seven- to ten-fold increase in clotting activity and a concomitant decrease in apparent molecular weight. The latter was comparable for each activation system yielding the following average molecular weight values: factor VaSa, 246,000-, factor Va RVV-V, 251,500; Factor Vathr, 239,000; alpha-chymotrypsin, but not trypsin, can activate plasma factor V yielding a product similar to that observed with the above activators. The molar quantities of each of the activators required varied considerably with thrombin having the highest specific activity and factor Xa the lowest. Activation by factor Xa was greatly facilitated by the addition of phospholipid. In the presence of a mixture of phosphatidylcholine/phosphatidylserine (1:1, w/w), the activation of factor V by factor Xa plus Ca2+ required one-third the amount of factor Xa protein as that required in the absence of phospholipid. Even though each of these activators appears to act in an enzymatic manner, the chemical nature of the conversion is unknown at this time.     

Factor IX San Dimas. Substitution of glutamine for Arg-4 in the propeptide leads to incomplete gamma-carboxylation and altered phospholipid binding properties

DNA sequence analysis of the Factor IX gene from a hemophilia B patient (98% Factor IX antigen; less than 0.01 unit/ml clotting activity) has identified a point mutation in exon II. A guanine to adenine transition causes the substitution of a glutamine codon for an arginine codon at -4 in the propeptide of Factor IX. This variant, termed Factor IX San Dimas, circulates in the plasma as proFactor IX with a mutant 18-amino acid propeptide still attached. Like Factor IX Cambridge (Arg-1----Ser), Factor IX San Dimas is unable to express metal-induced epitopes recognized by conformation-specific polyclonal antibodies. Amino acid analysis of the alkaline hydrolysate indicates that purified Factor IX San Dimas contains a reduced number of gamma-carboxyglutamyl residues compared to Factor IX. However, this protein undergoes metal-induced quenching of the intrinsic fluorescence. In addition, Factor IX San Dimas is unable to interact with phospholipid vesicles. The absence of coagulant activity in Factor IX San Dimas can be attributed to impaired calcium-induced conformational changes and loss in the ability to bind phospholipid vesicles in the presence of calcium ions.     

The role of phosphatidylglycerol in the activation of CTP:phosphocholine cytidylyltransferase from rat lung.

The reaction catalyzed by CTP:phosphocholine cytidylyltransferase in the reverse direction, i.e. the formation of CTP and phosphocholine from CDP-choline and pyrophosphate, is slightly faster than the reaction in the forward direction. The reverse reaction is optimal at 2 mM pyrophosphate and 6 mM Mg2+, in both fetal and adult preparations. The apparent substrate Km values for phosphocholine, CDP-choline, and pyrophosphate are similar in the fetal and adult forms of the enzyme. The enzyme activity is separated into two forms by gel filtration. The enzyme from adult lung exists as a high molecular weight species, ranging in size from 5 X 10(6) to 50 X 10(6). The enzyme from fetal lung exists as a 190,000 molecular weight species and is totally dependent upon added anionic phospholipid for activity in both the forward and reverse direction. The addition of phosphatidylglycerol gives maximal activity, while phosphatidylinositol or cardiolipin produce about 60 to 70% of the maximal activity. Enzyme activation is accompanied by an aggregation of the enzyme. A sonicated preparation of phosphatidylglycerol is a more efficient activator than a preparation mixed on a Vortex mixer (KA = 30 micronM) and also converts a larger proportion of enzyme from fetal lung into a high molecular weight species. The enzyme from adult lung can be dissociated into a form in fetal lung. The dissociated species can be converted back to a high molecular weight form in the presence of phosphatidylglycerol.     

Preparation and properties of derivatives of bovine factor X and factor Xa from which the gamma-carboxyglutamic acid containing domain has been removed

Limited proteolysis of bovine blood coagulation Factor X by chymotrypsin produces a derivative in which the light chain is cleaved between Tyr 44 and Lys 45. Two peptide products, residues 1-44 of the Factor X light chain and a modified zymogen, Factor X(-GD) have been isolated and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, elution behavior on anion-exchange chromatography, amino acid composition, and by partial amino acid sequence determination. Factor X(-GD) no longer contains the 12 gamma-carboxyglutamic acid residues of the native zymogen and thus serves as a model for investigation of the properties conferred on Factor X by the presence of gamma-carboxyglutamic acid. Cleavage of Factor X at Tyr 44 by chymotrypsin is inhibited by Ca2+ and Mg2+ ions. Factor X(-GD) is activated by the coagulation factor activator of Vipera russellii venom, but at less than 1% of the rate of activation of native Factor X. The susceptibility of Tyr 44 to chymotryptic cleavage implies that this residue is on the surface of the light chain of Factor X. Factor Xa(-GD) is indistinguishable from native Factor Xa in its activity on Benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide, on prothrombin alone, and on prothrombin plus Factor Va. In the presence of phospholipid the rate of prothrombin activation catalyzed by Factor Xa(-GD) is the same as in the absence of phospholipid.     

Purification and characterization of Ricinus communis invertase

An invertase from Ricinus communis leaves was purified 4,400-fold. The preparation was homogeneous by criteria of gel electrophoresis, gel permeation, adsorption, and ionic exchange chromatography. One optimum pH at 3.5 was observed with crude invertase; however, purified preparations showed two optima, at pH 3.5 and 5.5. Addition of bovine serum albumin restored one maximum at pH 3.5 and elicited a 30% activation of the invertase. The effect was caused by many other proteins and by heparin, dextran sulfate, and polyvinylpyrrolidone. Fructose, fructose 1,6-diphosphate, maleic, trans-aconitic, malic, and ascorbic acids were simple competitive inhibitors of the purified enzyme. Glucose was a noncompetitive inhibitor. The activation by proteins suppressed these inhibitory effects. The minimum concentration of activator necessary to reach the maximal activation or "point of optimal activation" was always reached at a concentration of 1 X 10(-6) M, independently of the nature of the activator, when 8.6 X 10(-12) mol of enzyme were used. Apparent molecular weight determinations of the enzyme in the presence and absence of activator and molecular weight determinations based on determinations of the point of optimal activation suggested that the purified enzyme is a heptamer (Mr of 77,900, Stokes radius 32 A, frictional ration f/fo 1.1, partial specific volume 0.749 ml/g) and that the activated form is a trimer consisting of two enzyme subunits and one activator molecule. The activation was lost by dilution of the trimer. The enzyme subunit, as isolated by gel filtration in the presence of sodium dodecyl sulfate (Mr 11,000) was inactive but quickly regained activity upon removal of sodium dodecyl sulfate.     

Human thrombins. Production, evaluation, and properties of alpha-thrombin.

Human alpha-thrombin, the thromboplastin activation product of prothrombin with high clotting and esterase activity, was produced from Cohn Fraction III paste. The procedure started with 0.4 to 3.2 kg of frozen paste and was completed in 2 or 3 days. Some 23 g of thrombin were recorded for 65 quantitated preparations made from 11 lots of Fraction III paste. These preparations were obtained at protein concentrations of 3.9 +/- 1.3 mg/ml with a yield of 340 +/- 110 mg/kg of paste, which represented 48 +/- 14% of the clotting potential extracted as prothrombin. They had specific clotting activities of 2.8 +/- 0.4 U.S. (NIH) units/microng of protein and titrated to 88 +/- 8% active with p-nitrophenyl-p'-guanidinobenzoate (NPGB). Those (N - 29) examined by labeling with [14C]diisopropyl phosphorofluoridate (iPr2P-F) and electrophoresing in sodium dodecyl sulfate (SDS)-polyacrylamide gels were found to contain only (N = 4) or predominantly alpha-thrombin (97 +/- 3%) and corresponding amounts of ists degradation product, beta-thrombin (2.6 +/- 3.1%). No plasmin(ogen), prothrombin complex factors (II, VII, IX, IXalpha, X, Xalpha), or prothrombin fragments were detected in representative preparations. As produced in 0.75 M NaCl, pH approximately 6, thrombin was stable for approximately 1 week at 4 degrees and for greater than 1 year at less than or equal to 50 degrees; freeze-dried thrombin stored at 4 degrees for greater than 1 year displayed stable clotting activity and no vial to vial variation, permitting its use for reference purposes. Human thrombin generated by Taipan snake venom activation was compared with that produced by rapid thromboplastin activation: after treatment with [14C]iPr2P-F, greater than 95% of the label in both thrombins migrated at the same rate during electrophoresis in SDS; identical pairs of NH2-terminal residues were released in three consecutive Edman degradation cycles.     

The contributions of Ca2+, phospholipids and tissue-factor apoprotein to the activation of human blood-coagulation factor X by activated factor VII.

In the extrinsic pathway of blood coagulation, Factor X is activated by a complex of tissue factor, factor VII(a) and Ca2+ ions. Using purified human coagulation factors and a sensitive spectrophotometric assay for Factor Xa, we could demonstrate activation of Factor X by Factor VIIa in the absence of tissue-factor apoprotein, phospholipids and Ca2+. This finding allowed a kinetic analysis of the contribution of each of the cofactors. Ca2+ stimulated the reaction rate 10-fold at an optimum of 6 mM (Vmax. of 1.1 x 10(-3) min-1) mainly by decreasing the Km of Factor X (to 11.4 microM). In the presence of Ca2+, 25 microM-phospholipid caused a 150-fold decrease of the apparent Km and a 2-fold increase of the apparent Vmax. of the reaction; however, both kinetic parameters increased with increasing phospholipid concentration. Tissue-factor apoprotein contributed to the reaction rate mainly by an increase of the Vmax., in both the presence (40,500-fold) and absence (4900-fold) of phospholipid. The formation of a ternary complex of Factor VIIa with tissue-factor apoprotein and phospholipid was responsible for a 15 million-fold increase in the catalytic efficiency of Factor X activation. The presence of Ca2+ was absolutely required for the stimulatory effects of phospholipid and apoprotein. The data fit a general model in which the Ca2(+)-dependent conformation allows Factor VIIa to bind tissue-factor apoprotein and/or a negatively charged phospholipid surface resulting into a decreased intrinsic Km and an increased Vmax. for the activation of fluid-phase Factor X.     

Binding of human blood-coagulation Factors IXa and X to phospholipid membranes.

A simple centrifugation technique has been developed to study the interaction of human coagulation Factors IXa and X with phospholipid membranes. In the presence of Ca2+, equimolar phosphatidylserine/phosphatidylcholine membranes form tight complexes with Factor X (KD = 2.8 X 10(-8) M); the KD is independent of the phospholipid concentration. Binding sites are available for about 2 mmol of Factor X/mol of phospholipid. Factor IXa has a slightly higher affinity for the phospholipid membrane (KD = 1.2 X 10(-8)M), and competes with Factor X for binding. The experimentally observed competition between Factor X and Factor IXa is in agreement with a model that describes the binding of two distinct ligands to a single class of independent binding sites.     

The contribution of Ca2+ and phospholipids to the activation of human blood-coagulation Factor X by activated Factor IX.

The role of the cofactors Ca2+ and phospholipid in the activation of human Factor X by Factor IXa was investigated. By use of a sensitive spectrophotometric Factor Xa assay, it was demonstrated that human Factor IXa can activate Factor X in the absence of cofactors. The presence of Ca2+ as the only cofactor resulted in a 7-fold stimulation of the Factor Xa formation. Kinetic analysis of the Ca2+-stimulated reaction showed that the apparent Km of Factor X was 4.6 microM, whereas the apparent Vmax. for Factor Xa formation was 0.0088 mol of Xa/min per mol of IXa. The presence of phospholipid as the only cofactor had no effect on the rate of Factor Xa formation. However, a several-hundred-fold stimulation was observed when Ca2+ and phospholipid were present in combination. The activation of Factor X in the presence of Ca2+ and phospholipid was found to be kinetically heterogeneous, involving both phospholipid-bound and free reactants. Quantitative data concerning the phospholipid binding of Factors IXa and X were used to study the relation between the rate of Factor Xa formation and the binding of enzyme and substrate to the phospholipid membrane. The results support the hypothesis that phospholipid-bound Factor X is the substrate in the phospholipid-stimulated reaction; however, phospholipid-bound and free Factor IXa seem to be equally efficient in catalysing the activation of phospholipid-bound Factor X.     

Activation and control of factor VII by activated factor X and thrombin. Isolation and characterization of a single chain form of factor VII.

Factor VII purified as previously described, was found to consist of two polypeptide chains joined by disulfide bridges. We now report the isolation and 200,000-fold purification of a single chain form of Factor VII. This was accomplished by protecting the molecule against proteolysis by including benzamidine during the entire purification. The purification was essentially as previously reported except that barium cirtate was substituted for barium sulfate as an absorbant for Factor VII as it resulted in a 4-fold increase in yield. Single chain Factor VII is rapidly hydrolyzed by Factor Xa in the presence of calcium ions and phospholipids, and by thrombin, to a two-chain form which possesses at least 85 times the Factor VII clotting activity of the single chain species. The two-chain form of the enzyme requires tissue factor in order to activate Factor X. From the observed rates of activation of Factor VII by Xa in the presence of clacium ions and phospholipids, it was claculated that at approximately physiological concentration, Factor VII activity would increase at an initial rate of 20-fold per min; this reaction is sufficiently rapid to constitute a feedback control mechanism. The action of thrombin is approximately 40-fold slower under these conditions. Diisopropylphosphorofluoridate inactivates the single chain and two-chain forms of Factor VII at approximately equal rates. After inhibition, the single chain species could be cleaved but not activated by proteolysis.     

Preparation of bovine blood coagulation factors X1 and X2

A method is described for the preparation of both Factor X1 and Factor X2 from citrated bovine blood. The proteins from the plasma were first adsorbed on barium citrate by adding barium chloride solution. The precipitate formed was stirred with citrate/NaOH pH 6.9 buffer; barium and other clotting factors were removed by adding ammonium sulphate (up to 30% saturation) to the suspension. The Factor X was then precipitated by 65% ammonium sulphate, after resolution in citrate buffer chromatographed on DEAE-Sephadex and purified by rechromatography on DEAE-Sephadex and DEAE-Sepharose, respectively. This yielded Factor X1 and Factor X2 with respective purifications of about 16 000 and 24 000-fold that of the plasma. The apparent molecular mass of both Factor X1 and Factor X2 was 55 kDa as estimated by the sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Factor X2 had a higher specific biological activity of about 340 000 units/mg compared to that of Factor X1 of about 230 000 units/mg.     

Further evidence for the concept of bovine plasma arylesterase as a lipoprotein.

Purified preparations of bovine plasma arylesterase were obtained by isoelectric focusing of enzyme prepared by (NH4)2SO4 fractionation of plasma and chromatography on DEAE-cellulose and Sephadex G-200. Although the high-density-lipoprotein fraction (HDL2) of serum provides an alternative source of enzyme, the enzymic activity of preparations made from it is much less stable. The purified arylesterase preparation has a molecular weight of 440000 and a partial specific volume of 0.91 ml/g, properties indistinguishable from those of the less highly purified enzyme. Extraction with acetone and ether removes neutral lipids from the enzyme, but the resulting lipid-depleted preparation retains most of the phospholipid present initially. A partial specific volume of 0.81 ml/g and a minimum molecular weight of approx. 100000 were determined for the lipid-depleted preparations of arylesterase. The present results support the concept of bovine plasma arylesterase as a lipoprotein in its own right, rather than as an enzymic polypeptide that is loosely associated with the HDL2 fraction of serum.     

Residual Factor VIII-like cofactor activity of thioredoxin and related oxidoreductases

Background

Factor VIII is the cofactor for Factor X activation by Factor IXa. Activated Factor X, Factor Xa, in turn activates prothrombin in a sequence that leads to fibrin clot formation at the site of vascular injury. Although the biochemistry of the cascade has been well studied, the molecular mechanism underlying the cofactor role of Factor VIII is not understood.

Methods

We screened a bacterial peptide display library with Factor IXa and Factor X co-immobilized on tosylactivated Dynabeads which were then used as platelet surrogates. Validation of peptide selection procedure and comparison of Factor VIII-like cofactor activity of oxidoreductases was performed using COATEST assays. Determination of Factor VIII as a folding catalyst with potential disulphide isomerase activity was determined using the RNase A renaturation assay.

Results

We set out to identify the cofactor requirements of the Factor IXa/Factor X procoagulant complex by random peptide display, and isolated a peptide with the active-site sequence, CGPC, of thioredoxin. This peptide was able to activate Factor X in a Factor IXa-dependent manner. Redox catalysts or oxidoreductases with homologous active-site vicinal cysteines such as PDI and DsbA also mimicked Factor VIII in their requirement of Factor IXa in Factor X activation. However, the cofactor activity of these peptides was up to a 1000-fold lower than that of Factor VIII and they were therefore unable to catalyse blood coagulation. Factor X activation by PDI and by Factor VIII was abolished by oxidation in an isolated system, which implies a possible role for thiol–disulphide exchange in the activity of the tenase complex. Using scrambled RNase A as a surrogate substrate, we also found that Factor VIII could renature this enzyme.

Conclusion

Our findings suggest that Factor VIII may be a specialized folding catalyst with disulphide isomerase activity. We suggest that it is this activity that may underlie its cofactor function in Factor X activation, and that this function is interchangeable with classical oxidoreductases.

General significance

The possible involvement of thiol–disulphide interchange as a mechanism underlying Factor VIII cofactor activity may provide some insight into the biochemistry of the intrinsic tenase complex.     

Characterization of the fibrin polymer structure that accelerates thrombin cleavage of plasma factor XIII

The effect of plasmin-derived fibrin(ogen) degradation products on alpha-thrombin cleavage of plasma Factor XIII was studied to identify the fibrin polymer structure that promotes Factor XIIIa formation. Fibrin polymers derived from fibrinogen and Fragment X enhanced the rate of thrombin cleavage of plasma Factor XIII in plasma or buffered solutions. The concentrations of fibrinogen and Fragment X that promoted half-maximal rates of Factor XIIIa formation were 5 and 40 micrograms/ml, respectively. Fragments Y, D, E, D-dimer, and photooxidized fibrinogen did not enhance thrombin cleavage of Factor XIII. Although purified Fragment D1 inhibited fibrin gelation, the soluble protofibrils promoted thrombin activation of Factor XIII. Noncrosslinked fibrin fibers failed to enhance thrombin cleavage of Factor XIII. In conclusion, soluble fibrin oligomers function to promote thrombin cleavage of plasma Factor XIII during blood clotting.     

A light-scattering study of bovine factor VIII

Classical light-scattering measurements made on bovine Factor VIII preparations, which contain a series of multimers ranging from 1 greater than 12 X 10(6) molecular weight, gave a weight average molecular weight of about 8 X 10(6) and a z-average radius of gyration of about 3000 A. Characteristic dimensions for variously shaped models were calculated.     

Leukocyte complement: interleukin-like properties of factor Bb

It has been previously shown that the activated form of Factor B (Factor Bb) of the alternative pathway of complement activation stimulates monocyte spreading and killing of xenogenic erythrocytes and staphylococci. Factor Bb also stimulates lymphocyte blastogenesis in vitro, and native (uncleaved) Factor B is a major constitutive product of murine macrophages. To evaluate the possible "monokine" or "lymphokine"-like properties of Factor Bb, a radioimmunoassay was developed to measure the quantities of Factor B in phytohemagglutinin (PHA)-mitogen-stimulated cultures of human peripheral blood mononuclear cells. Nonstimulated mononuclear cell cultures from human peripheral blood (containing 10-14% monocytes and greater than 85% lymphocytes) at a density of 3 X 10(6) cells/ml (in serum-free medium) released less than 7 X 10(-10) M/liter (60 ng/ml) of Factor B antigen in 24 hr at 37 degrees C, and when mononuclear cells were stimulated with PHA mitogen in serum-free medium, the levels of Factor B antigen in media at 24 hr were significantly higher 1-3 X 10(-8) M/liter (0.9-2.8 micrograms/ml). The molecular size of Factor B in these media was 50-65 kDa by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a size appropriate for Factor Bb (60 kDa). Since pathological effects of macrophages in autoimmune disease may result from the release of lysosomal hydrolases, the effects of purified Factor Bb on mononuclear phagocytes were investigated in an in vitro system of murine peritoneal exudate macrophages. Factor Bb induced secretion of marker lysosomal hydrolases N-acetyl-beta-D-glucosaminidase (hexosaminidase) and beta-glucuronidase from thioglycollate-elicited murine peritoneal exudate macrophages in a dose-response and kinetic manner. Hydrolase release was induced in serum-free medium without a known particulate activator at a concentration of 80-200 nM (5-13 micrograms/ml) Factor Bb. Maximal release occurred in 3-5 hr at 37 degrees C and extracellular enzyme activity of hexosaminidase and glucuronidase increased as intracellular enzyme levels decreased, suggesting that Factor Bb triggers release of these enzymes from intracellular lysosomal pools. These results provide an example of a complement protein which is synthesized, released, and activated during mononuclear cell culture and which induces release of lysosomal enzymes from macrophages. In conventional terminology, Factor B or Factor Bb might be termed a "lymphokine," "monokine," or "interleukin".     

Blood clotting factor IX Niigata: substitution of alanine-390 by valine in the catalytic domain

Factor IX Niigata is a mutant factor IX responsible for the moderately severe hemophilia B in a patient who has a normal level of factor IX antigen with reduced clotting activity (1-4% of normal). We reported previously that the purified mutant protein could be converted to the factor IXa beta form by factor XIa/Ca2+ at a rate similar to that in the case of normal factor IX, but the resulting mutant factor IXa beta could not activate factor X in the presence of factor VIII, Ca2+, and phospholipids (Yoshioka, A. et al. (1986) Thromb. Res. 42, 595-604). In the present study, we analyzed factor IX Niigata at the structural level to elucidate the molecular abnormality responsible for the loss of clotting activity. Amino acid sequence analysis of a peptide obtained on lysyl endopeptidase digestion, coupled with subsequent SP-V8 digestion, demonstrated that the alanine at position 390 was substituted by valine in the catalytic domain of the factor IX Niigata molecule.     

CDP-diglyceride:inositol transferase from rat liver. Purification and properties.

CDP-diglyceride:inositol transferase, which catalyzes the final step of the de novo synthesis of phosphatidylinositol, was solubilized by sodium cholate from microsomes prepared from rat liver and purified by ammonium sulfate fractionation, sucrose density gradient centrifugation, and DEAE-cellulose column chromatography. Addition of phospholipid during the purification and the assay procedures prevented irreversible loss of the enzyme activity to some extent. The resulting preparation was nearly homogeneous as judged by polyacrylamide gel electrophoresis. The recovery of the purified enzyme from the microsomal fraction was 3 to 3.3% with respect to activity and 0.12% with respect to amount of protein. The molecular weight of the enzyme was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 60,000. The purified enzyme required exogenous phospholipds for its activity. Various phospholipid classes activated the enzyme rather nonspecifically. The Km for myo-inositol was 2.5 X 10(-3) M and that for CDP-diglyceride was 1.7 X 10(-4) M. The pH optimum was 8.6. The enzyme required Mm2+ or Mg2+ for activity. The optimal concentration of Mn2+ for activation was 0.5 mM, while the activity in the presence of Mg2+ increased up to 20 mM. The enzyme was inhibited by thiol-reactive reagents. There was a competition for inositol by inosose-2 but not by scyllitol.     

Combination of affinity chromatography and analytical polyacrylamide gel electrophoresis for rapid measurement of human serum high-density lipoprotein apoproteins

Heparin of an average molecular weight of 13,000 was fractionated on the basis of size into five fractions of different weight-average molecular weight ranging from 8500 to 20,000. The heparin was also degraded using microbial heparinase resulting in products ranging from a disaccharide of molecular weight 500 to an oligosaccharide of molecular weight 3100. These products were also size fractionated. The individual heparin fractions and products were tested for metachromatic activity with Azure A. The metachromatic activity of the heparin fractions was independent of molecular weight, while the metachromatic activity of the products was dependent on molecular weight. Metachromatic activity was found in a fragment as small as a tetrasaccharide. Anticoagulant activity was found in fragments of tetrasaccharide or larger by a Factor Xa clotting assay and in fragments of hexasaccharide or larger by a Factor Xa amidolytic chromogenic assay.