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Query: UMLS:C0027960 (mole)
 21,279 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A study was undertaken to investigate the influence of fasting (24 hours), epinephrine (four 0.25 mg/kg s.c. doses at hourly intervals), adrenocorticotropin (two 40 I.U. s.c. doses at 2-hour intervals) and immobilization stress (4 hours) on the response of rats to some coumarin or indanedione anticoagulants. The anticoagulants were always administered first and were followed immediately or within the next hour by the appropriate challenge. Fasting produced a significant enhancement of the antiprothrombin response to warfarin (0.5 mg/kg i.v. or 0.75-3.0 mg/kg s.c.), bishydroxycoumarin (7.5 mg/kg i.p. or 10 mg/kg s.c.) and phenindione (40 mg/kg p.o). Epinephrine and immobilization stress, but not adrenocorticotropin, similarly prolonged the prothrombin time after warfarin (0.75-3.0 mg/kg s.c.). When used in the absence of anticoagulants, all challenges had no effect on the prothrombin time. In addition, fasting did not affect the response of anticoagulated animals to vitamin K. Plasma free fatty acids were significantly increased by the various challenges. The binding constant of warfarin to undiluted plasma proteins were decreased from the control value by a factor of 1.7 and 2.0 as a result of immobilization stress and fasting, respectively. Fasting per se increased the amount of bound endogenous free fatty acids per mole of protein; the latter parameter was further increased in the presence of warfarin. The present data show that fasting and stress enhance the anticoagulant response to warfarin and suggest that this might be due to an interference of endogenous free fatty acids with binding of warfarin to plasma proteins.
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PMID:The influence of fasting and stress on the response of rats to warfarin. 5 16

Factor V and factor Va binding to single bilayer phospholipid vesicles was investigated by light-scattering intensity measurements. This technique allows the measurement of free and phospholipid-bound protein concentrations from which equilibrium constants can be obtained. As controls, the Ca2+-dependent phospholipid binding of prothrombin and factor X were also studied. The average values obtained for the dissociation constants (Kd) and lipid to protein ratio at saturation, moles/mole (n), for prothrombin (Kd = 2.3 X 10(-6) M, n = 104) and factor X (Kd = 2.5 X 10(-6) M, n = 46) binding to vesicles containing 25% Folch fraction III and 75% phosphatidylcholine in the presence of 2 mM Ca2+ were in agreement with those reported in the literature. The average factor V and factor Va values for the dissociation constants and lipid to protein ratio at saturation (moles/mole) were Kd = 7.2 X 10(-8) M and n = 270 for factor V and Kd = 4.4 X 10(-7) M and n = 76 for factor Va. In contrast to prothrombin and factor X, factor V and factor Va demonstrated Ca2+-independent lipid binding. In addition, the number of factor V and factor Va molecules bound per vesicle was found to be dependent both on the phosphatidylserine content of the vesicle and the ionic strength of the buffer.
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PMID:Phospholipid-binding properties of bovine factor V and factor Va. 48 30

A procedure for the preparation of highly purified pig prothrombin is described. Compared to the initial clotting activity of the starting plasma, this protein was purified 776 times with a final yield of 8 per cent. The purified zymogen showed a specific activity of 1,460 NH units/mg of protein , a molecular weight of 65,000 as determined by SDS-polyacrylamide disc gel electroesis, E 1.0 mg/ml 1.0 cm, 280 nm = 1.45 at pH 7.0 and the following amino acid composition: Asx 51, Thr 38, Glx 62, Pro 23, Gly44, Ala 25, Half-Cys 30, Val 35, Met 3, Ile 30, Leu 32, Tyr 19, Phe 22, Lys 36, His 8, Arg 28, and Trp 13, which accounts for a minimum molecular weight of 59,370 (carbohydrates not computed). Alanine was found as the only N-terminal residue. Carboxypeptidases A and B failed to release any C-terminal residue. By hydrazinolysis however 0.4 mole of serine was released per mole of prothrombin. The activation of crude and chromatographed pig prothrombin was investigated.
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PMID:Pig prothrombin: purification and properties. 95 54

Coagulation Factor VII from bovine plasma is a glycoprotein containing a single peptide chain. The NH2-terminal sequence of Ala-Asx-Gly-Phe-Leu- is homologous with the NH2 termini of prothrombin, Factor IX, and the light chain of Factor X. Factor Xa in the presence of calcium ions and phospholipid cleaves Factor VII at an Arg-Ile bond in the sequence Arg-Ile-Val-Gly-Gly-, producing a two-chain molecule with at least 85 times the coagulant activity of single-chain Factor VII and a new NH2-terminal sequence homologous with the corresponding chains of thrombin, Factor IXa and Factor Xa. A second slower cleavage at an Arg-Gly bond destroys Factor VII activity. Bovine Factor VII, unlike prothrombin, Factor IX, and Factor X, is rapidly inhibited by diisopropylphosphorofluoridate (iPr2PF). [3H]iPr2PF is readily incorporated into one-chain, two-chain, and three-chain forms of Factor VII up to ratios of approximately 0.9 moles of [3H]diisopropylphosphate per mole of protein. The radioactive peptides generated from each form of [32P]iPr2PF-inhibited Factor VII by tryptic and thermolytic digestion were found to migrate together on paper electrophoresis. This indicates that the iPr2PF is incorporated stoichiometrically into the same specific site in each form.
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PMID:Mechanism of activation of bovine factor VII. Products of cleavage by factor Xa. 95 65

The stability, buoyancy, and intrinsic activity for factor V-phospholipid complexes were investigated. The decay of factor V activity in the absence of phospholipid followed first order kinetics; however, in the presence of several phospholipids a biphasic decay curve was observed. The addition of phosphatidylethanolamine to factor V produced only a small loss of activity in the first 2 minutes but decreased the subsequent rate of inactivation fourfold. A PE-factor V complex with a low bouyant density was separated from uncomplexed factor V by sucrose density ultracentrifugation. The association constant for this complex was 5 X 10(6) M-1 with approximately 2 moles of factor V bound per mole of lipid micelle. The isolated complex was capable of increasing prothrombin conversion 10-fold without additional phospholipid. A still lighter complex increased the rate of prothrombin conversion 18-fold. Phosphatidyl serine produced a concentration-dependent loss of up to 95% of the factor V activity in the first 2 minutes. After ultracentrifugation on a sucrose density gradient, a PS-factor V complex of increased density was detected. This complex failed to accelerate prothrombin conversion in the intrinsic two-stage assay. Except at very high concentrations, phosphatidylcholine did not alter the kinetics of inactivation of factor V. A factor V-phosphatidylcholine complex could not be detected after ultracentrifugation. When added to factor V, cardiolipin (200 mug/ml), produced a rapid 50% decline in activity with a subsequent three-fold increase in the rate of inactivation. No activity was recovered after ultracentrifugation of factor V in the presence of cardiolipin. Saturated phosphatidylethanolamine produced a concentration dependent initial loss of activity, but only a minimal increase in the subsequent rate of inactivation. At 200 mug/ml almost no light complex was detected after ultracentrifugation, but at 800 mug/ml a light complex was observed. This behavior corresponds to the ability of saturated phosphatidylethanolamine to accelerate prothrombin conversion only at very high concentrations. Thus, phospholipids combine with factor V to form complexes which differ in their ability to accelerate prothrombin conversion. The most active species are stable lipoprotein complexes of lower buoyant density than factor V.
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PMID:Determinants of the formation and activity of factor V-phospholipid complexes. II. Molecular properties of the complexes. 118 12

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)
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PMID:Effect of membrane fluidity and fatty acid composition on the prothrombin-converting activity of phospholipid vesicles. 139 Jul 58

The multivariant approach offers best possibilities for assessment of liver function. The role of the different clinical, clinico-laboratory and combined clinical and clinicochemical indices in the prognosis of liver cirrhosis was studied in patient in ambulatory conditions. A step regressive mathematical model with the help of the program 2R of the statistical package BMDP was used. The regression of the clinical indices by 5 steps of the mathematical model showed that of greatest importance for the survival are the following indices: ascites, months since its onset, collaterals, anorexia and vascular nevi. By 4 steps of the regressive model of the clinico-chemical indices the following indices were chosen: prothrombin time, albumin, total bilirubin, cholesterol and alkaline phosphatase. The regression of the combined clinical and clinico-chemical indices pointed out as basic factors 3 clinical indices (ascites, months since its onset, collaterals) and 3 clinico-chemical indices related to the disturbed liver function (prothrombin time, total bilirubin, albumin).
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PMID:[Evolution and prognosis in patients with liver cirrhosis. II. A multifactorial analysis using a stepped regression mathematical model]. 208 Jun 13

Human prothrombin and prothrombin fragment 1 were demonstrated to bind to Phenyl-TSK columns in the presence of 5.0 mM calcium ions but not in the presence of either magnesium ions or manganese ions. The calcium-dependent interaction of prothrombin fragment 1 is markedly reduced upon oxidation of approximately one mole of tryptophan per mole of protein. The ability of prothrombin fragment 1 to inhibit prothrombin activation by factor Xa in the presence of calcium ions and phospholipid is also markedly reduced by reaction with N-bromosuccinimide. These results provide the first demonstration of a calcium-specific site in prothrombin outside of the "GLA domain".
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PMID:A hydrophobic site in human prothrombin present in a calcium-stabilized conformer. 319 39

A method is presented for analysis of gamma-carboxyglutamic acid based on its derivatization with phenylisothiocyanate and reverse phase HPLC analysis of the resulting phenylthiocarbamyl derivative. Proteins were hydrolyzed with sodium hydroxide and the hydrolysates were desalted on Dowex 50 eluted with ammonium hydroxide. The resulting amino acid mixtures were derivatized with phenylisothiocyanate and the phenylthiocarbamyl derivatives were separated under isocratic conditions on either C18 or C8 reverse phase columns using 0.14 M Tris, 0.05% triethylamine, titrated to pH 7.5 with glacial acetic acid, plus 2% acetonitrile, and detected by absorbance at 254 nm. The method is linear over the range from 10 to 1000 pmol of gamma-carboxyglutamic acid and the limit of detection is near 2 pmol. The utility of the method was verified for analysis of purified prothrombin yielding a value of 10.3 mol of gamma-carboxyglutamic acid per mole in agreement with sequence data. No gamma-carboxyglutamate was detectable for acid-hydrolyzed samples of prothrombin, nor in acid- or base-hydrolyzed samples of bovine serum albumin. Application of this method failed to corroborate the reported presence of gamma-carboxyglutamate in a putative mitochondrial gamma-carboxyglutamate-containing calcium-binding protein. The method was also tested for determination of beta-carboxyaspartate, beta-hydroxyaspartate, phosphoserine, phosphothreonine, and phosphotyrosine in an attempt to identify an unknown material which appeared in preparations of the mitochondrial protein.
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PMID:Analysis of gamma-carboxyglutamic acid by reverse phase HPLC of its phenylthiocarbamyl derivative. 326 14

The prothrombin activator from the venom of Oxyuranus scutellatus (Taipan snake) was purified by gel filtration on Sephadex G-200 and ion-exchange chromatography on QAE-Sephadex. The activator is a large protein with a molecular weight of approximately 300,000, which is composed of subunits of Mr 110,000 and 80,000 and two disulfide-linked polypeptides of Mr 30,000. One or both of these Mr 30,000 subunits contain the active site. The venom activator readily converts Factor Xa-specific chromogenic substrates and is also able to activate prothrombin (Km = 166 microM, Vmax = 2.5 mumol of prothrombin activated per min/mg of venom). Gel electrophoretic analysis of prothrombin activation indicates that the venom activator randomly cleaves the Arg274-Thr275 and Arg323-Ile324 bonds of prothrombin since both thrombin and meizothrombin are formed as reaction products. Venom-catalyzed prothrombin activation is not affected by bovine Factor Va but is greatly stimulated by phospholipids plus Ca2+ ions. This stimulatory effect is explained by a decrease of the Km for prothrombin. In the presence of 50 microM phospholipid vesicles (25% phosphatidylserine/75% phosphatidylcholine; mole/mole), the Km is 0.34 microM and the Vmax is 7.1 mumol of prothrombin activated per min/mg of venom. The purified venom activator contains gamma-carboxyglutamic acid residues which presumably function in the interaction between the venom activator and phospholipids. Treatment of the activator with 0.8 M NaSCN strongly reduces its ability to activate prothrombin but has no effect on its amidolytic activity. The prothrombin-converting activity of the NaSCN-treated activator can be restored with bovine Factor Va. During prolonged gradient gel electrophoresis, the Mr 300,000 activator dissociates into smaller subunits. This causes a loss of the prothrombin-converting activity, while the amidolytic activity is recovered in a protein with an apparent molecular weight of 57,000. This protein can, however, rapidly activate prothrombin in the presence of Factor Va or in the presence of a protein component of Mr 220,000 that also migrates on the gel. These results suggest that the prothrombin activator from the O. scutellatus venom is a multimeric protein complex consisting of a Factor Xa-like enzyme and a Factor Va-like cofactor.
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PMID:Prothrombin activation by an activator from the venom of Oxyuranus scutellatus (Taipan snake). 353 Nov 98


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