Mathematical model of the activation of prothrombin by factor X a and factor V t

A mathematical model of prothrombin activation is being proposed which includes the feedback mechanism of thrombin and the alteration of factor V by thrombin. This model is in good agreement with experimental data for the dependence of the rate of thrombin formation on the concentrations of factors V and X _a . In particular, it correctly predicts the existence and location of a maximum in both of these cases.     

Importance of lysine-286 at the NADP site of glutamate dehydrogenase from Salmonella typhimurium.

Affinity labeling studies of NADP(+)-glutamate dehydrogenase from Salmonella typhimurium have shown that the peptide Leu-282-Lys-286 is located near the coenzyme site [Haeffner-Gormley et al. (1991) J. Biol. Chem. 266, 5388-5394]. The present study was undertaken to evaluate the role of lysine-286. The mutant enzymes K286R, K286Q, and K286E were prepared by site-directed mutagenesis, expressed in Escherichia coli, and purified. The Vmax values (micromoles of NADPH per minute per milligram of protein) were similar for WT (270), K286R (529), K296Q (409), and K286E (382) enzymes. As measured at pH 7.9, the Km value for NADPH was much greater for K286E (280 microM) than for WT (9.8 microM), K286R (30 microM), or K286Q (66 microM) enzymes. The efficiencies (kcat/Km) of the WT and K286R mutant were similar (1.2 x 10(3) min-1 microM-1 and 1.0 x 10(3) min-1 microM-1, respectively) while those of K286Q (0.30 x 10(3) min-1 microM-1) and K286E (0.07 x 10(3) min-1 microM-1) were greatly reduced. The decreased efficiency of the K286E mutant results from the increase in Km-NADPH, consistent with a role for a basic residue at position 286 which enhances the binding of NADPH. Plots of Vmax vs pH showed the pH optima to be 8.1-8.3 for all enzymes at saturating NADPH concentrations. A 40-fold increase in Km-NADPH for K286E was observed as the pH increased from 5.98 to 8.08, from which a unique pKe of 6.5 was calculated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of vitamin D3 metabolites in Callithrix jacchus (common marmoset)

Vitamin D₃ (cholecalciferol) is endogenously produced in the skin of primates when exposed to the appropriate wavelengths of ultraviolet light (UV-B). Common marmosets (Callithrix jacchus) maintained indoors require dietary provision of vitamin D₃ due to lack of sunlight exposure. The minimum dietary vitamin D₃ requirement and the maximum amount of vitamin D₃ that can be metabolized by marmosets is unknown. Observations of metabolic bone disease and gastrointestinal malabsorption have led to wide variation in dietary vitamin D₃ provision amongst research institutions, with resulting variation in circulating 25-hydroxyvitamin D₃ (25(OH)D₃), the accepted marker for vitamin D sufficiency/deficiency. Multiple studies have reported serum 25(OH)D₃ in captive marmosets, but 25(OH)D₃ is not the final product of vitamin D₃ metabolism. In addition to serum 25(OH)D_3, we measured the most physiologically active metabolite, 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), and the less well understood metabolite, 24,25-dihydroxyvitamin D₃ (24,25(OH)₂D₃) to characterize the marmoset's ability to metabolize dietary vitamin D₃. We present vitamin D₃ metabolite and related serum chemistry value colony reference ranges in marmosets provided diets with 26,367 (Colony A, N = 113) or 8,888 (Colony B, N = 52) international units (IU) of dietary vitamin D₃ per kilogram of dry matter. Colony A marmosets had higher serum 25(OH)D₃ (426 ng/ml [SD 200] vs. 215 ng/ml [SD 113]) and 24,25(OH)₂D₃ (53 ng/ml [SD 35] vs. 7 ng/ml [SD 5]). There was no difference in serum 1,25(OH)₂D₃ between the colonies. Serum 1,25(OH)₂D₃ increased and 25(OH)D₃ decreased with age, but the effect was weak. Marmosets tightly regulate metabolism of dietary vitamin D₃ into the active metabolite 1,25(OH)₂D₃; excess 25(OH)D₃ is metabolized into 24,25(OH)₂D₃. This ability explains the tolerance of high levels of dietary vitamin D₃ by marmosets, however, our data suggest that these high dietary levels are not required.     

Export of proteins from oocytes of Xenopus laevis.

When human lymphoblastoid mRNA was microinjected into X. laevis oocytes, titers of interferon rapidly reached a maximum inside the oocyte while accumulation of interferon continued in the incubation medium for at least 45 hr. If interferon protein was injected into oocytes it was rapidly inactivated. Significantly, newly synthesized interferon but not injected interferon was found to be membrane-associated. Further experiments involving the co-injection of mRNAs coding for secretory proteins (guinea pig milk proteins and human interferon) and nonsecretory proteins (rabbit globin) revealed that only the secretory proteins were exported from the oocyte. Moreover, different proteins were exported at different rates. A distinct subclass of newly synthesized oocyte proteins of unknown function also accumulated in the incubation medium. Since the information encoded in the messenger RNAs of secretory proteins is sufficient to specify synthesis, compartmentation and secretion of these proteins, the oocyte may provide a complete system for the analysis of the secretory process.     

Antigenic structure and variation in an influenza virus N9 neuraminidase.

We previously determined, by X-ray crystallography, the three-dimensional structure of a complex between influenza virus N9 neuraminidase (NA) and the Fab fragments of monoclonal antibody NC-41 [P. M. Colman, W. G. Laver, J. N. Varghese, A. T. Baker, P. A. Tulloch, G. M. Air, and R. G. Webster, Nature (London) 326:358-363, 1987]. This antibody binds to an epitope on the upper surface of the NA which is made up of four polypeptide loops over an area of approximately 600 A2 (60 nm2). We now describe properties of NC-41 and other monoclonal antibodies to N9 NA and the properties of variants selected with these antibodies (escape mutants). All except one of the escape mutants had single amino acid sequence changes which affected the binding of NC-41 and which therefore are located within the NC-41 epitope. The other one had a change outside the epitope which did not affect the binding of any of the other antibodies. All the antibodies which selected variants inhibited enzyme activity with fetuin (molecular weight, 50,000) as the substrate, but only five, including NC-41, also inhibited enzyme activity with the small substrate N-acetylneuramin-lactose (molecular weight, 600). These five probably inhibited enzyme activity by distorting the catalytic site of the NA. Isolated, intact N9 NA molecules form rosettes in the absence of detergent, and these possess high levels of hemagglutinin activity (W.G. Laver, P.M. Colman, R.G. Webster, V.S. Hinshaw, and G.M. Air, Virology 137:314-323, 1984). The enzyme activity of N9 NA was inhibited efficiently by 2-deoxy-2,3-dehydro-N-acetylneuraminic acid, whereas hemagglutinin activity was unaffected. The NAs of several variants with sequence changes in the NC-41 epitope lost hemagglutinin activity without any loss of enzyme activity, suggesting that the two activities are associated with separate sites on the N9 NA head.     

The Effect of pH, O(2), and Temperature on the CO(2) Compensation Point of Isolated Asparagus Mesophyll Cells

The effect of pH, O₂ concentration, and temperature on the CO₂ compensation point (Г[CO₂]) of isolated Asparagus sprengeri Regel mesophyll cells has been determined in a closed, aqueous environment by a sensitive gas-chromatographic technique. Measured values range between 10 and 100 microliters per liter CO₂ depending upon experimental conditions. The Г(CO₂) increases with increasing temperature. The rate of increase is dependent upon the O₂ concentration and is more rapid at high (250-300 micromolar), than at low (30-60 micromolar), O₂ concentrations. The differential effect of temperature on Г(CO₂) is more pronounced at pH 6.2 than at pH 8.0, but this pH-dependence is not attributable to a direct, differential effect of pH on the relative rates of photosynthesis and photorespiration, as the O₂-sensitive component of Г(CO₂) remains constant over this range. The Г(CO₂) of Asparagus cells at 25°C decreases by 50 microliters per liter when the pH is raised from 6.2 to 8.0, regardless of the prevailing O₂ concentration. It is suggested that the pH-dependence of Г(CO₂) is related to the ability of the cell to take up CO₂ from the aqueous environment. The correlation between high HCO₃⁻ concentrations and low Г(CO₂) at alkaline pH indicates that extracellular HCO₃⁻ facilitates the uptake of CO₂, possibly by increasing the flux of inorganic carbon from the bulk medium to the cell surface. The strong O₂− and temperature-dependence of Г(CO₂) indicates that isolated Asparagus mesophyll cells lack an efficient means for concentrating intracellular CO₂ to a level sufficient to reduce or suppress photorespiration.     

Characterization of an active site peptide modified by the substrate analogue 3-bromo-2-ketoglutarate on a single chain of dimeric NADP+-dependent isocitrate dehydrogenase

The substrate analogue 3-bromo-2-ketoglutarate reacts with pig heart NADP+-dependent isocitrate dehydrogenase to yield partially inactive enzyme. Following 65% inactivation, no further inactivation was observed. Concomitant with this inactivation, incorporation of 1 mol of reagent/mol of enzyme dimer was measured. The dependence of the inactivation rate on bromoketoglutarate concentration is consistent with reversible binding of reagent (KI = 360 microM) prior to irreversible reaction. Manganous isocitrate reduces the rate of inactivation by 80% but does not provide complete protection even at saturating concentrations. Complete protection is obtained with NADP+ or the NADP+-alpha-ketoglutarate adduct. By modification with [14C]bromoketoglutarate or by NaB3H4 reduction of modified enzyme, a single major radiolabeled tryptic peptide was obtained by high performance liquid chromatography with the sequence: Asp-Leu-Ala-Gly-X-Ile-His-Gly-Leu-Ser-Asn-Val-Lys. Evidence in the following paper (Bailey, J.M., Colman, R.F. (1987) J. Biol. Chem. 262, 12620-12626) indicates that X is glutamic acid. Enzyme modified at the coenzyme site by 2-(bromo-2,3-dioxobutylthio)-1,N(6)-ethenoadenosine 2',5'-biphosphate in the presence of manganous isocitrate is not further inactivated by bromoketoglutarate. Bromoketoglutarate-modified enzyme exhibits a stoichiometry of binding isocitrate and NADPH equal to 1 mol/mol of enzyme dimer, half that of native enzyme. These results indicate that bromoketoglutarate modifies a residue in the nicotinamide region of the coenzyme site proximal to the substrate site and that reaction at one catalytic site of the enzyme dimer decreases the activity of the other site.     

Isolation of the glutamyl peptide labeled by the nucleotide analogue 2-(4-bromo-2,3-dioxobutylthio)-1,N(6)-ethenoadenosine 2',5'-biphosphate in the active site of NADP+-specific isocitrate dehydrogenase

2-(4-Bromo-2,3-dioxobutylthio)-1,N(6)-ethenoadenosine 2',5'-bisphosphate (2-BDB-T epsilon A-2',5'-DP) is an affinity label for the coenzyme-binding site of pig heart NADP+-dependent isocitrate dehydrogenase. Specific reaction occurs at the coenzyme site with an incorporation of 0.5 mol of reagent/mol of enzyme subunit (i.e. modification of only one subunit of the dimeric enzyme) (Bailey, J.M., and Colman, R.F. (1985) Biochemistry 24, 5367-5377). Modified enzyme, prepared by incubating 1 mg/ml isocitrate dehydrogenase with 75 microM 2-BDB-T epsilon A-2',5'-DP in the absence and presence of substrate or coenzyme, was reduced with NaBH4, carboxymethylated, and digested with trypsin. Nucleotidyl peptides were isolated by chromatography on DEAE-cellulose, followed by treatment with acid phosphatase (to decrease the negative charge by removing the phosphate groups from covalently bound reagent) and rechromatography on the same DEAE-cellulose column. The isolated peptides were characterized by amino acid analysis, dansylation, and gas-phase sequencing. A single triskaidekapeptide corresponding to modification of the coenzyme site by 2-BDB-T epsilon A-2',5'-DP was identified as: Asp-Leu-Ala-Gly-X-Ile-His-Gly-Leu-Ser-Asn-Val-Lys. Additional evidence indicated that X is a glutamate residue derivatized by 2-BDB-T epsilon A-2',5'-DP.     

Cleavage of a 100 kDa membrane protein (aggregin) during thrombin-induced platelet aggregation is mediated by the high affinity thrombin receptors

Thrombin-induced platelet aggregation is accompanied by cleavage of aggregin, a surface membrane protein (Mr = 100 kDa), and is mediated by the intracellular activation of calpain. We now find that agents that increase intracellular levels of platelet cAMP by stimulating adenylate cyclase, also inhibit thrombin binding and platelet activation by destabilizing thrombin receptors on the platelet surface. Iloprost (a stable analog of PGI2) and forskolin each completely inhibited platelet aggregation by 2 nM thrombin and markedly decreased cleavage of aggregin. Thrombin inactivated by D-phenylalanine-L-prolyl-L-arginine chloromethyl ketone (PPACK-thrombin) binds to the highest affinity site for thrombin on the platelet surface, but thrombin modified by N alpha-tosyl-L-lysine chloromethylketone (TLCK-thrombin) does not. We now demonstrate that preincubation of platelets with PPACK-thrombin blocked platelet aggregation and cleavage of aggregin induced by 2 nM thrombin. In contrast, TLCK-thrombin neither blocked platelet aggregation nor the cleavage of aggregin. These results show that a) platelet aggregation and cleavage of aggregin by thrombin (2nm) involves the occupancy of high affinity alpha-thrombin receptors on the platelet surface, and b) stimulators of adenylate cyclase which increase cAMP, inhibit thrombin-induced platelet aggregation and cleavage of aggregin by mechanisms which include inhibiting the binding of thrombin to its receptors.     

The sequence HGLGHGHEQQHGLGHGH in the light chain of high molecular weight kininogen serves as a primary structural feature for zinc-dependent binding to an anionic surface.

The histidine-glycine-rich region of the light chain of cleaved high molecular weight kininogen (HK) is thought to be responsible for binding to negatively charged surfaces and initiation of the intrinsic coagulation, fibrinolytic, and kinin-forming systems. However, the specifically required amino acid sequences have not been delineated. An IgG fraction of a monoclonal antibody (MAb) C11C1 to the HK light chain was shown to inhibit by 66% the coagulant activity and by 57% the binding of HK to the anionic surface of kaolin at a concentration of 1.5 microM and 27 microM, respectively. Proteolytic fragments of HK were produced by successive digestion with human plasma kallikrein and factor XIa (FXIa). Those polypeptides that bound tightly (Kd = 0.77 nM) to a C11C1 affinity column were eluted at pH 3.0 and purified by membrane filtration. On 15% SDS polyacrylamide electrophoresis, the approximate M(r) was 7.3 kDa (range 6.2-8.1 kDa). Based on N-terminal sequencing, this polypeptide (1(2)), which extends from the histidine residue 459 to a lysine at position 505, 509, 511, 512, 515, or 520, inhibits by 50% the coagulant activity expressed by HK at a concentration of 22 microM. The synthetic peptide HGLGHGH representing the N-terminal of the 1(2)) fragment was synthesized, tested, and found at 4 mM to inhibit the procoagulant activity of HK 50%. A synthetic heptadecapeptide, HGLGHGHEQQHGLGHGH (residues 459-475) included within the 1(2) fragment, and with the ability to bind zinc, inhibited 50% of the HK coagulant activity at a concentration of 325 microM in the absence and presence of added Zn2+ (30 microM). The specific binding of 125I-HK to a negatively charged surface (kaolin) was inhibited 50% by unlabeled HK (5 microM). HGLGHGH, at a concentration of 7.0 mM, inhibited the binding to kaolin by 50%. The heptadecapeptide inhibited the specific binding of 125I-HK to kaolin by 50%, at a concentration of 2.3 mM, in the absence of Zn2+. In contrast, when Zn2+ was added, the concentration to achieve 50% inhibition decreased to 630 microM, indicating that Zn2+ was required to attain a favorable conformation for binding. Moreover, the 1(2) fragment was found to inhibit 50% of the 125I-HK binding to kaolin at a concentration of 380 microM. These results suggest that residues contained within the 1(2) fragment, notably HGLGHGHEQQHGLGHGH, serves as a primary structural feature for binding to a negatively charged surface.