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101.
A steady-state kinetic analysis of the activation of bovine Factor X, by bovine Factor Xa, was undertaken. The activation was found to be dependent on the presence of divalent cations; Ca2+ showing the greatest stimulatory effect and Mn2+ exhibiting a lower degree of activity for this reaction. Although Sr2+ and Mg2+ were ineffective when present alone, each contributed synergistically to the activation rate at suboptimal levels of Ca2+. The effect of phospholipid (phosphatidylcholine:phosphatidylserine, 4:1, w:w) on the rate of activation and on the activation pathway was investigated. Phospholipid (PL) concentrations of up to 40 μm had no effect on the activation rate; whereas, concentrations of 40–180 μm were slightly inhibitory. In the absence of PL, the major product of the activation was Factor α-Xa, while in the presence of PL, lower-molecular-weight forms of Factor X (Factor β-X) and Factor Xa (Factor β-Xa were produced. At saturating levels of Ca2+, the Km app for the activation, at pH 7.4 and 37 °C, in the absence of PL, was found to be 0.6 ± 0.1 μm and the V was 1.7 ± 0.3 mol Factor X cleaved min?1 mol?1 Factor Xa. The corresponding values, in the presence of 90 μm PL, were 1.4 ± 0.2 μm and 2.2 ± 0.2 mol Factor X cleaved min?1 mol?1 Factor Xa. 相似文献
102.
Activation of wheat chloroplast sedoheptulose bisphosphatase: a continuous spectrophotometric assay 总被引:3,自引:0,他引:3
A new continuous spectrophotometric assay for sedoheptulose 1,7-bisphosphatase, applied to studies of the activation and steady-state kinetics of the wheat enzyme, is described. The assay enzyme sequence couples the formation of sedoheptulose 7-phosphate to the oxidation of NADH. The recycling of the reaction substrate enables measurements to be made at essentially constant substrate concentrations. Activation of wheat chloroplast sedoheptulose 1,7-bisphosphatase required a reducing agent and could be described by a first-order rate constant. The rate of activation was greatly increased in the presence of Mg2+ and sedoheptulose 1,7-bisphosphate. The Km of the activated enzyme for sedoheptulose 1,7-bisphosphate. and its S0.5 for Mg2+ were found to be 13.3 μm and 1.6 mm respectively. A high recovery method for purifying wheat chloroplast sedoheptulose 1,7-bisphosphatase is also detailed. 相似文献
103.
The hen oviduct shell gland is a highly active calcium-transporting epithelial tissue which is responsible for the mineralization of the egg shell. We have identified a calcium-stimulated ATPase present at high specific activity in membrane preparations from shell gland mucosal shavings. In the presence of optimal MgCl2 (5 mm) and a Ca2+ buffer, ATP hydrolysis was stimulated by addition of low concentrations of free Ca2+ (K0.5 ~0.4 μm); but not by similar concentrations of Mn2+, Zn2+, Co2+, or La2+. This stimulation was specific for ATP; there was little or no effect of Ca2+ on hydrolysis of ADP, AMP, GTP, ITP, or p-nitrophenyl phosphate. Calcium-stimulated ATPase activity was inhibited by chlorpromazine, trifluoperazine, and quercetin, as well as by sulfhydryl-blocking agents, but not by oligomycin or ouabain. No significant effect of calmodulin was observed. Finally, low concentrations of free Ca2+ (10 to 100 μm) in the presence or absence of Mg2+ stimulated transfer of 32P from [γ-32P]ATP to a 105,000 molecular weight shell gland membrane protein. This phosphoprotein was sensitive to hydrolysis by heating or by hydroxylamine treatment at acidic pH, and its formation was not inhibited by addition of K+. The specific activity of Ca2+-ATPase in total membrane preparations from laying hen shell gland ranged from 80 to 150 nmol/min/ mg protein, similar to or greater than levels found in purified plasma membrane fractions from a variety of tissues. No significant activity was found in membrane preparations from the magnum or isthmus regions of the oviduct, which are not involved in egg shell calcification. The characteristics of the Ca2+-ATPase, its high specific activity, and its preferential localization in the shell gland region of the oviduct suggest a role for an ATP-dependent calcium transport system in egg shell mineralization. 相似文献
104.
The detergent solubilization of dog kidney (Na + K)-ATPase has been investigated. The nonionic detergents, Brij 58, C12E8, and Lubrol WX were tested for their ability to produce active, soluble enzyme. Lubrol WX gave the best results. Enzyme so treated is found in the supernatant fraction after centrifugation at 100,000g for 1 h. It has the same or slightly greater specific activity, the same subunit composition as judged by SDS-gel electrophoresis, and very similar kinetic parameters with respect to sodium, potassium, ATP, pNPP, and ouabain as the membrane-bound enzyme. The Lubrol-treated enzyme is stable for at least 5 days at 4 °C. The phospholipid content of the Lubrol-treated enzyme is decreased, as might be expected, by about 50%. Limited tryptic proteolysis and fluorescence changes seen after modification with FITC indicate that the solubilized (Na + K)-ATPase undergoes the same conformational transitions as the membrane enzyme. Our results indicate that kidney enzyme solubilized as described here is nondenatured and fully active, and therefore a valuable preparation for spectroscopic and other approaches for study of this enzyme. 相似文献
105.
Defect in 3'-phosphoadenosine 5'-phosphosulfate synthesis in brachymorphic mice. II. Tissue distribution of the defect 总被引:2,自引:0,他引:2
The tissue distribution of the defective PAPS synthetic pathway in homozygous brachymorphic mice () has been investigated using four different criteria: (i) incorporation of 35SO42? into adenosine 5′-phosphosulfate (APS), 3′-phosphoadenosine 5′-phosphosulfate (PAPS), and endogenous macromolecular acceptors, (ii) APS kinase (adenylylsulfate kinase; ATP:adenylylsulfate 3′-phosphotransferase, EC 2.7.1.25) activity, (iii) ATP sulfurylase (sulfate adenylyltransferase; ATP:sulfate adenylyltransferase, EC 2.7.7.4) activity, (iv) thermostability of ATP sulfurylase. With respect to the first three criteria, the results indicate that liver is affected as profoundly as cartilage (K. Sugahara and N. B. Schwartz, Arch. Biochem. Biophys. (1982) 214, 589–601). In contrast, skin and brain show no differences between normal and mutant. Kidney is significantly, but only moderately, affected. The results from thermostability studies demonstrate that ATP sulfurylase activity is more labile in cartilage, liver, and kidney, but not in skin or brain, supporting the above-observed distribution of the defect. Therefore, the present results indicate a multiple, but not universal, tissue distribution of the defective PAPS synthetic pathway in mice. Furthermore, these findings support the suggestion that ATP sulfurylase as well as APS kinase is defective in brachymorphic mice. 相似文献
106.
Cyclic GMP-dependent protein kinase has been purified to apparent homogeneity from bovine adrenal cortex and its presence in the rat adrenal cortex has been demonstrated. Sucrose density sedimentation studies indicated that the Mr of the enzyme was 145,000. This protein was composed to two identical subunits each with Mr of 75,000. The enzyme molecule was asymmetric with a frictional coefficient of 1.54, Stokes radius of 53.5 Å and a sedimentation coefficient of 6.5. The enzyme self-phosphorylated and the stoichiometry of cyclic GMP binding was two molecules per holoenzyme. Calmodulin or troponin C markedly stimulated the apparent maximal velocity of cyclic GMP-dependent protein kinase without affecting its basal activity. This effect of protein modulators was independent of calcium. Sucrose density gradient studies indicated that the stimulatory effect of calmodulin was due to its interaction with histones. An interaction of calmodulin with the enzyme was not observed. The steroidogenic potential of cyclic GMP and its analogs correlated closely with their ability to stimulate cyclic GMP-dependent protein kinase; the order of potency for both activities was 8-bromocylic GMP > cyclic GMP > N2-monobutyryl cyclic GMP > N2, O2-dibutyryl cyclic GMP. In each case, calmodulin enhanced the cyclic GMP-dependent protein kinase activity for histone phosphorylation. These results indicate that although cyclic GMP is the primary regulator of cyclic GMP-dependent protein kinase, other modulator proteins such as calmodulin could act as additional regulators of the phosphorylation of substrate proteins. In addition, the demonstration of cyclic GMP-dependent protein kinase in rat adrenal glands, and the results with cyclic GMP and its analogs relating to their activation of protein kinase and steroidogenesis are consistant with the concept that cyclic GMP is one of the mediators of adrenal steroidogenesis. 相似文献
107.
G J Howlett P W Dickson H Birch G Schreiber 《Archives of biochemistry and biophysics》1982,215(1):309-318
The molecular weights of a number of 125I-labeled plasma proteins have been determined from an analysis of their sedimentation equilibrium behavior in an air-driven ultracentrifuge. The values obtained agree well with results obtained by other methods. Molecular weights obtained for 125I-labeled bovine serum albumin and the rat serum proteins albumin, α1-acid glycoprotein, and major acute-phase α1-protein were unaffected by the addition of 7% rat plasma. Direct evidence for protein-protein interactions was obtained for mixtures of 125I-labeled rat α1-acid glycoprotein and the plant lectin concanavalin A and for mixtures of 125I-labeled protein A from Staphylococcus aureus and 7% rat plasma. Interactions of a different type were observed when the sedimentation equilibrium profiles of 125I-labeled proteins were determined in concentrated solutions of other proteins. Under these conditions the effects of molecular exclusion or nonideality became significant and low estimates were obtained for the molecular weights of the labeled proteins. Analysis of the data obtained for 125I-labeled bovine serum albumin in concentrated solutions of bovine serum albumin (20–80 mg/ ml) yielded nonideality coefficients in good agreement with literature values. Analysis of the behavior of 125I-labeled rat serum albumin, transferrin, and α1-acid glycoprotein yielded nonideality coefficients and hence activities of these proteins in undiluted rat plasma. 相似文献
108.
Permeabilization of nitrogen-starved cells of Escherichia coli W with Lubrol WX leads to a selective inactivation of the uridylyl-removing uridylyltransferase (UR/ UTase) enzyme of the glutamine synthetase (GS) cascade system; whereas similar treatment does not affect activity of UR/UTase in cells grown under conditions of nitrogen excess (10 mm glutamine) (Mura, U., and Stadtman, E. R. (1981) J. Biol. Chem.256, 13014–13021). The possibility that susceptibility to Lubrol inactivation is related to differences in the state of adenylylation of GS and/or in the state of uridylylation of the PII protein was investigated. Permeabilized cells from nitrogen sufficient as well as from nitrogen-limited growth medium were exposed to Lubrol after prior incubation under conditions that lead to high or low states of GS adenylylation and high or low PIID/PIIA ratios. Integrity of UR/UTase was monitored by measuring the capacity of UTP to stimulate the deadenylylation of GS in situ. The results showed that the inactivation of UR/UTase by Lubrol is not affected by the states of GS adenylylation or PII uridylylation. 相似文献
109.
Antibodies directed toward human erythrocyte Ca2+-ATPase: effect on enzyme function and immunoreactivity of Ca2+-ATPases from other sources 总被引:3,自引:0,他引:3
Antibodies directed against purified human erythrocyte Ca2+-ATPase (purified according to a procedure modified from V. Niggli, J. T. Penniston, and E. Carafoli, 1979, J. Biol. Chem., 254, 9955–9958) were raised in rabbits. In competitive radioimmunoassay tests of immunological cross-reactivity, human erythrocyte Ca2+-ATPase shows a consistent pattern of immunological similarity to the Ca2+-ATPases derived from cell surface fractions of other species, such as rat and dog erythrocyte ghosts, rat corpus luteum plasma membranes, and rat brain synaptic plasma membranes. On the other hand, a purified Ca2+-ATPase preparation from rabbit skeletal muscle sarcoplasmic reticulum failed to show any immunological similarity to the human enzyme. The amount of Ca2+-ATPase protein in the erythrocyte ghosts was estimated to be about 0.6 μg/mg ghost protein, which was not too different from the calculated value of 1.2 ± 0.2 μg/mg ghost protein (mean ± SD, n = 6) based on the calmodulin binding studies of the erythrocyte ghosts. Anti-Ca2+-ATPase immunoglobulin G inhibited enzyme activity and calcium transport, showing that at least one subpopulation of antibodies can block the active site of the enzyme. The antibodies had no effect on the binding of calmodulin to erythrocyte membranes. 相似文献
110.
Hepatic ATP-citrate lyase prepared with a fluoride-free step to allow endogenous phosphatases to dephosphorylate the enzyme was phosphorylated in vitro by the catalytic subunit of cyclic AMP-dependent protein kinase and [γ-32P]ATP. After electrophoresis the radioactive phosphate was located predominantly in the gel slice containing the Coomassie blue stained protein corresponding to ATP-citrate lyase. The Stoichiometry of phosphorylation of hepatic ATP-citrate lyase in vitro by the catalytic subunit was such that 0.53 ± 0.02 molecules of phosphate were incorporated per subunit. The degree of phosphorylation was independent of the amount of ATP-citrate lyase present as substrate in the concentration range 1.2–6.4 μm. In the absence of catalytic subunit there was very little labeled phosphate incorporated into ATP-citrate lyase. Phosphorylation of ATP-citrate lyase by catalytic subunit was abolished by the specific protein inhibitor of cyclic AMP-dependent protein kinase. When ATP-citrate lyase was subjected to electrophoresis under nondenaturing conditions, lyase activity was recovered from the gel slice corresponding to the Coomassie blue staining phosphoprotein of a stained gel run in parallel. 相似文献