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1.
Dithionite reduced azoferredoxin and molybdoferredoxin from Clostridium pasteurianum W5 were oxidatively titrated with various electron acceptors. The AzoFd gave up 0.87 electrons per AzoFd monomer (27,500 mol. wt.). The oxidation reduction potential of AzoFd, determined by equilibrium with redox dyes, was ?0.240 V. Dithionite reduced MoFd gave up 3.6 electrons per MoFd tetramer (220,000 mol. wt.). The oxidation reduction potential for MoFd was ?0.070 V. Because the potential of this MoFd half cell is so positive, the electrons removed during this oxidation may not be those that reduce dinitrogen.  相似文献   

2.
The 2′,3′-dialdehyde of ADP, obtained by periodate oxidation of ADP, inhibited the hydrolytic activity of the purified Ca2+, Mg2+-activated ATPase of Escherichiacoli. In the initial stages of the reaction inhibition was due to the reaction of 1 mol inhibitor/active site. When non-specific labelling of amino groups by the dialdehyde was lowered by the simultaneous presence of 15 mM ATP in the reaction mixture, 3 mol “ATP-protectable” binding sites/mol ATPase were found. “ATP-protectable” binding of the dialdehyde was not observed when the hydrolytically inactive ATPase of an unc A mutant of E.coli was used although binding of the inhibitor to non-protected amino groups still occurred. This suggests that the mutant ATPase is unable to bind ATP or that the amino groups with which the dialdehyde reacts in the native enzyme are absent or masked.  相似文献   

3.
Chromatophores from Rhodospirillum rubrum contain 12 × 10?3 mol ATP and 8.3 × 10?3 mol ADP per mol chlorophyll, tightly bound to the coupling ATPase. Under energised conditions, these exchange slowly with added nucleotide. Using single turnover light flashes, it is demonstrated that the release of bound ATP is too slow to be on the direct pathway of photophosphorylation.  相似文献   

4.
D Desaiah  L K Cutkomp  R B Koch 《Life sciences》1973,13(12):1693-1703
The ATPase enzyme system from two-spotted spider mites, Tetranychusurticae (Koch) was sensitive, in vitro, to four acaricides. Tricyclohexylhydroxytin (PlictranR) was an outstanding inhibitor of oligomycin-sensitive (mitochondrial) Mg2+ATPase from fish brain and spider mite homogenates. The I50 values were 6.6×10?11M and 6.2×10?10M, respectively. Less effective were chlorbenside, chlorfenethol and ovotran. Plictran at a higher concentration (2×10?7M) was also more effective on Na+-K+ATPase both in mites and fish brain homogenates as compared to chlorfenethol, chlorbenside and ovotran. Plictran inhibited oligomycin-insensitive Mg2+ATPase at concentrations of 10?8M but stimulated at high concentrations (5×10?6M and higher).  相似文献   

5.
Cholinergic synaptic vesicles isolated from the electric organ of Torpedo californica are confirmed to exhibit energy-linked uptake of [3H] acetylcholine. [3H]Acetylcholine is concentrated in the vesicles by a factor of 10–14 in the presence of MgATP and bicarbonate. This active uptake can be completely inhibited by the mitochondrial uncouplers 3-t-butyl-5-Cl-2′-Cl-4′-nitro-salicylanilide (S-13) and p-nitrophenol. The vesicle-associated ATPase is stimulated by S-13 in the same concentration range which inhibits [3H]acetylcholine active uptake. The ATPase also is stimulated by valinomycin. Both S-13 and valinomycin effects are independent of exogenous Ca2+. Thus, a proton gradient generated by the vesicle-associated ATPase appears to be coupled to active [3H]acetylcholine uptake.  相似文献   

6.
Calmodulin-like activity in the soluble fraction of Escherichia coli   总被引:8,自引:0,他引:8  
A heat-stable factor with properties similar to those of calmodulin was found in the fraction containing Ca2+-dependent cyclic AMP phosphodiesterase of Escherichiacoli. The factor activated such enzymes as cyclic nucleotide phosphodiesterase of bovine brain, (Ca2+,Mg2+)ATPase of human erythrocyte menbrane and myosin light chain kinase of rabbit myometrium in a Ca2+-dependent fashion with an apparent Ka of 5 × 10?5M. The factor and brain calmodulin had no effect on the phosphodiesterase of E.coli. It may be concluded that calmodulin or a calmodulin-like protein occurs in prokaryotes.  相似文献   

7.
Perfused rat hearts were treated with isoprenaline (10?6M) or ouabain (5.5 × 10?6M). The phosphate contents of troponin-I and myosin P light chains were established by radiolabelling with 32P; in the case of the light chains, direct chemical analysis of total and of specifically alkali-labile phosphate was also performed. Addition of isoprenaline caused phosphorylation of both troponin-I and myosin P light chains, reaching a maximum increment, after several minutes, of 1 mol/mol and 0.30 mol/mol, respectively. The Mg2+-ATPase activities, at saturating Ca2+ concentrations, of natural actomyosin isolated from treated hearts were significantly depressed, and an inverse correlation was established between the phosphate content of troponin-I and the Vmax[Ca2+] of this ATPase activity. The Ca2+ sensitivity of the Ca2+Mg2+-ATPase was also decreased. These changes were all reversed by an incubation permitting dephosphorylation of proteins by endogenous phosphatases.Treatment of hearts with ouabain caused no increment in troponin-I phosphorylation, but increased the P light chain phosphate content to a maximum of 0.30 mol/mol after some minutes. A positive correlation was evident between phosphate content of the light chains (in all experiments) and the maximum myosin Ca2+-ATPase activities. In addition, the Vmax[ATP] of the Ca2+Mg2+-ATPase of natural actomyosin was increased when light chain phosphorylation had occurred in the absence of troponin-I phosphorylation. P-light chain phosphorylation did not affect the Ca2+ sensitivity of Ca2+Mg2+-ATPase activity.We suggest that the effects of phosphorylation of troponin-I are to diminish thin filament sensitivity to Ca2+, and to decrease the efficiency of the transduction process along neighbouring actin monomers, such that the number of actin-myosin crossbridge interactions is decreased even in the presence of Ca2+ excess. Phosphorylation of P light chains of myosin has an activating effect on myosin Ca2+-ATPase activity, as well as on the rate of cross-bridge formation.  相似文献   

8.
Brinolase, a thrombolytic fungal protease capable of forming vasoactive kinins, has been shown to hydrolyze kinins after their formation. Using synthetic bradykinin as a substrate, the kinetics and mechanism of hydrolysis have been elucidated, evidently explaining the apparently low kinin formation in vivo, Bradykinin hydrolysis proceeded rapidly in vitro with a pH optimum of 7.0–7.5, and a half-life of 5.1 min, using 250 ng/ml bradykinin and 50 μg/ml brinolase. The Km was 3.2×10?6 M and the Vmax was 4.6 × 10?8 mol/liter/min, using 5 μg/ml brinolase. Two-dimensional paper fractionation of the brinolase-bradykinin digest revealed the presence of free arginine amongst the five peptide fragment spots.  相似文献   

9.
Methods are described for purification of a vesicular membrane fraction of hog gastric mucosa using differential centrifugation, density gradient separation on zonal rotors and free-flow electrophoresis. As a result a fraction is obtained enriched 40-fold in terms of K+-ATPase and free of any other enzyme marker other than K+-activated p-nitrophenyl phosphatase.the 5′-nucleotidase and basal Mg2+-ATPase are clearly separated from the latter enzymes.Osmotic shock, Triton X-100 treatment or K+ ionophores increased the K+-ATPase activity in isotonic conditions, but K+-p-nitrophenyl phosphatase is not affected by these treatments, nor is the ATPase activity in the presence of NH4+. The results suggest that the electrophoretic fraction contains a major population of tight vesicles, whose permeability to K+ is rate limiting for the ATPase activity but not for the p-nitrophenyl phosphatase activity. It is concluded that K+ site for the ATPase is internal whereas the K+ site for the p-nitrophenyl phosphatase is external, hence, the K+ site must be mobile across the membrane.  相似文献   

10.
A molybdenum-containing iron-sulfur protein has been isolated from the sulfate reducer Desulfovibrioafricanus. The protein appears to be a complex protein of high molecular weight (112,000) composed of 10 subunits (mol. wt. 11,500) and containing a high amount of molybdenum (5–6 atoms/mole) with approx. 20 atoms each of iron and labile sulfide. The spectrum shows peaks at around 615, 410 and 325 nm with a protein peak at 280 nm. Its millimolar extinction coefficients at 615, 410 and 280 nm are 48.4, 64.4 and 141 respectively. The protein contains 106 amino-acid residues per subunit of mol. wt. 11,262 and the number of cysteine residues is 2 per subunit. The N-terminal sequence which has been determined up to 26 residues is characterized by its high degree of hydrophobicity.  相似文献   

11.
The calmodulin activation of the (Ca2+ + Mg2+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) in human erythrocyte membranes was studied in the range of 1 nM to 40 μM of purified calmodulin. The apparent calmodulin-affinity of the ATPase was strongly dependent on Ca2+ and decreased approx. 1000-times when the Ca2+ concentration was reduced from 112 to 0.5 μM. The data of calmodulin (Z) activation were analyzed by the aid of a kinetic enzyme model which suggests that 1 molecule of calmodulin binds per ATPase unit and that the affinities of the calcium-calmodulin complexes (CaiZ) decreases in the order of Ca3Z >Ca4Z >Ca2Z ? CaZ. Furthermore, calmodulin dissociates from the calmodulin-saturated Ca2+-ATPase in the range of 10?7–10?6 M Ca2+, even at a calmodulin concentration of 5 μM. The apparent concentration of calmodulin in the erythrocyte cytosol was determined to be 3 to 5 μM, corresponding to 50–80-times the cellular concentration of Ca2+-ATPase, estimated to be approx. 10 nmol/g membrane protein. We therefore conclude that most of the calmodulin id dissociated from the Ca2+-transport ATPase in erythrocytes at the prevailing Ca2+ concentration (probably 10?7 – 10?8 M) in vivo, and that the calmodulin-binding and subsequent activation of the Ca2+-ATPase requires that the Ca2+ concentration rises to 10?6 – 10?5 M.  相似文献   

12.
Sensitivity of CaMg ATPase from axonic plasma membrane (APM) and sarcoplasmic reticulum (SR) of lobster, Homarus, americanus, to DDT was studied. The CaMg ATPase found in SR with the high Ca2+ affinity is sensitive to DDT while the portion of ATPase related to the low Ca2+ affinity site is not inhibited by DDT. Also, DDT is more inhibitory against the CaMg ATPase prepared from APM than the one obtained from SR. The relationship between inhibition of the CaMg ATPase by DDT in the axonic nerve membrane and in, vivo poisoning symptoms of the nervous system is discussed.  相似文献   

13.
Compound 4880, a condensation product of N-methyl-p-methoxyphenethylamine with formaldehyde, is composed of a family of cationic amphiphiles differing in the degree of polymerization. Compound 4880 was found to be a potent inhibitor of the calmodulin-activated fraction of brain phosphodiesterase and red blood cell Ca2+-transport ATPase, with IC50 values of 0.3 and 0.85 μg/ml, respectively. However, the basal activity of both enzymes is not at all suppressed by the drug at concentrations up to 300 μg/ml. Inhibition of Ca2+ transport into inside-out red blood cell vesicles by compound 4880 follows a similar pattern in that basal, calmodulin-independent, transport is also not affected by the drug. Kinetic analysis revealed that the stimulation of Ca2+-transport ATPase induced by calmodulin is inhibited by compound 4880 according to a competitive mechanism. It was demonstrated that the inhibitory constituents of compound 4880 bind to calmodulin in a Ca2+-dependent fashion. Comparison of the specificity of several anti-calmodulin drugs showed that compound 4880 is the most specific inhibitor of the calmodulin-dependent fraction of red blood cell Ca2+-transport ATPase that has been described hitherto. In addition, compound 4880 was found to be a rather specific inhibitor of the calmodulin-induced activation of Ca2+-transport ATPase when compared with the stimulation induced by an anionic amphiphile or by limited proteolysis. Half-maximal inhibition of the activity stimulated by oleic acid or mild tryptic digestion required 8- and 32-times higher concentrations of compound 4880, respectively, compared with the calmodulin-dependent fraction of the ATPase activity. Moreover, calmodulin-independent systems as rabbit skeletal muscle sarcoplasmic reticulum Ca2+-transport ATPase or calf cardiac sarcolemma (Na+ + K+)-transport ATPase are far less influenced by compound 4880 as compared with trifluoperazine and calmidazolium. Because of its high specificity compound 4880 is proposed to be a promising tool for studying calmodulin-dependent processes.  相似文献   

14.
Gastric microsomes do not contain any significant Ca2+-stimulated ATPase activity. Trypsinization of pig gastric microsomes in presence of ATP results in a significant (2–3-fold) increase in the basal (with Mg2+ as the only cation) ATPase activity, with virtual elimination of the K+-stimulated component. Such treatment causes unmaksing of a latent Mg2+-dependent Ca2+-stimulated ATPase. Other divalent cations such as Sr2+, Ba2+, Zn2+ and Mn2+ were found ineffective as a substitute for Ca2+. Moreover, those divalent cations acted as inhibitors of the Ca2+-stimulated ATPase activity. The pH optimum of the enzyme is around 6.8. The enzyme has a Km of 70 μM for ATP and the Ka values for Mg2+ and Ca2+ are about 4 · 10?4M and 10?7 M, respectively. Studies with inhibitors suggest the involvement of sulfhydryl and primary amino groups in the operation of the enzyme. Possible roles of the enzyme in gastric H+ transport have been discussed.  相似文献   

15.
DNA-binding proteins of Xenopus laevis synthesized during two periods of early development (oogenesis-ovulation and early embryogenesis) were co-chromatographed on DNA-cellulose. Proteins with an affinity for DNA were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Most of the proteins eluted from DNA-cellulose with 0.6 M NaCl had mol. wts less than 40 000; some of these proteins were synthesized to a greater extent by developing embryos than by oocytes. A DNA-binding protein or group of proteins with a mol. wt of approx. 70 000 was synthesized during oogenesis-ovulation but not during embryogenesis. Differential labeling of developing embryos with [3H]tryptophan and [14C]lysine indicated that some of the low mol. wt DNA-binding proteins are histones. Some of these proteins also incorporated monosodium [32P]phosphate. A greater fraction of the proteins synthesized by oocytes and developing embryos were bound to DNA-histone-cellulose than to DNA-cellulose. A group of low mol. wt proteins made during oogenesis-ovulation were bound more to DNA-histone-cellulose than were proteins with similar mol. wts made by developing embryos.  相似文献   

16.
Exposure of human erythrocyte ghosts (pH 8, 10°C) to visible light in the presence of the photosensitizer, methylene blue, results in a relatively rapid loss of spectrin (bands 1 and 2 on sodium dodecyl sulfate gel electropherograms) and the appearance of high molecular weight cross-linked derivatives. Isolated spectrin also undergoes photosensitized cross-linking, indicating that the reaction is not lipid-dependent.Extensive cross-linking was neither reversed by dithiothreitol nor prevented by prior blocking of SH groups with N-ethylmaleimide, suggesting that cysteine residues are not crucial bridging sites. The possible requirement for NH2 groups, as suggested by previous model studies (Dubbelman, T.M.A.R., de Goeij, A.F.P.M. and van Steveninck, J. (1978) Biochim. Biophys. Acta 511, 141–151), was tested. Succinylation of spectrin protected against cross-linking, but this effect is attributed to the disruption of quaternary structure, as deduced from sedimentation measurements. However, virtually complete blocking of NH2 groups by amidination perturbed overall structure relatively little, and had no effect on cross-linking. Moreover, exogenous amines such as ethylamine, added in large excess to spectrin prior to irradiation, did not interfere with cross-link formation. These results suggest that NH2 groups are not involved in the reaction.  相似文献   

17.
Fluorescein isothiocyanate was used to covalently label the gastric (H+ + K+)-ATPase. FITC treatment of the enzyme inhibited the ATPase activity while largely sparing partial reactions such as the associated p-nitrophenylphosphatase activity. ATP protected against inhibition suggesting the ligand binds at or near an ATP binding site. At 100% inhibition the stoichiometry of binding was 1.5 nmol FITC per mg Lowry protein a value corresponding to maximal phosphoenzyme formation. Binding occurred largely to a peptide of 6.2 isoelectric point, although minor labelling of a peptide of pI 5.6 was also noted. Fluorescence was quenched by K+, Rb+ and Tl+ in a dose-dependent manner, and the K0.5 values of 0.28, 0.83 and 0.025 mM correspond rather well to the values required for dephosphorylation at a luminal site. Vanadate, a known inhibitor of the gastric ATPase produced a slow Mg2+-dependent fluorescent quench. Ca2+ reversed the K+-dependent loss of fluorescence and inhibited it when added prior to K+. This may relate to the slow phosphorylation in the presence of ATP found when Ca2+ was substituted for Mg2+ and the absence of K+-dependent dephosphorylation. The results with FITC-modified gastric ATPase provide evidence for a conformational change with K+ binding to the enzyme.  相似文献   

18.
Hartmut Wohlrab  James Greaney 《BBA》1978,503(3):425-436
Mitochondria have been prepared from the flight muscles of mature blowflies (Sarcophaga bullata). Phosphate transport by these mitochondria, determined by rates of passive swelling in ammonium phosphate, is sensitive to inhibition by N-ethylmaleimide. 20 nmol of N-ethylmaleimide/nmol cytochrome a inhibit the swelling by 90%. When the mitochondria are inhibited by N-[3H]ethylmaleimide, then solubilized in dodecyl sulfate/mercaptoethanol at 100°C and then electrophoresed on dodecyl sulfate-polyacrylamide gels, many labeled protein bands can be detected, including a large labeled peak that has the same mobility as the tracking dye, bromophenol blue. Sonic submitochondrial particles that are prepared from the N-[3H]ethylmaleimidelabeled mitochondria, solubilized, and electrophoresed on dodecyl sulfatepolyacrylamide gels, possess only seven major labeled protein bands with no radioactive peak at the tracking dye. These labeled proteins have molecular weights of 71, 68, 64, 45, 32, 30, and approx. 10 · 103. The nmol N-[3H]-ethylmaleimide bound to each of these proteins per nmol cytochrome a are 0.15, 0.19, 0.35, 0.45, 0.87, 0.10, and 0.17, respectively, when the mitochondria are inhibited with 21.5 mol N-[3H]ethylmaleimide/mol cytochrome a at 10 μM cytochrome a. Coty and Pedersen ((1975) J. Biol. Chem. 250, 3515–3521) sensitized rat liver mitochondria to N-[3H]ethylmaleimide and identified five labeled proteins. Only the labeled 32 · 103 dalton and the 45 · 103 dalton proteins are common to both systems  相似文献   

19.
(1) The total phospholipid content of a gradient purified (K+ + H+)-ATPase preparation from pig gastric mucosa is 105 μmol per 100 mg protein, and consists of 29% sphingomyelin, 29% phosphatidylcholine, 28% phosphatidylethanolamine, 10% phosphatidylserine and 4% phosphatidylinositol. The cholesterol content corresponds to 50 μmol per 100 mg protein. (2) Treatment with phospholipase C (from Clostridium welchii and Bacillus cereus) results in an immediate decrease of the phosphate content. Up to 50% of the phospholipids are hydrolyzed by each phospholipase C preparation alone, without further hydrolysis by increased phospholipase concentration or prolonged incubation time. Combined treatment with the two phospholipase C preparations, sequentially or simultaneously, hydrolyzes up to 65% of the phospholipids. (3) The (K+ + H+)-ATPase and K+ stimulated p-nitrophenylphosphatase activities are decreased proportionally with the total phospholipid content, indicating that these enzyme activities are dependent on phospholipids. (4) Phospholipase C treatment does not change optimal pH, Km value for ATP and temperature dependence of the gastric (K+ + H+)-ATPase, but slightly decreases the Ka value for K+. (5) Phospholipase C treatment lowers the AdoPP[NH]P binding and phosphorylation capacities, suggesting that inactivation occurs primarily on the substrate binding level. (6) Most of the results can be understood by assuming that hydrolysis of the phospholipids by phospholipase C leads to aggregation of the membrane protein molecules and complete inactivation of the aggregated ATPase molecules.  相似文献   

20.
2,6-dibromothymoquinone (DBMIB) and other coenzyme Q analogs partially inhibit electron transport and the membrane-bound Mg++ stimulated ATPase of E. coli membranes. The inhibitions by DBMIB are fully reversed by coenzyme Q6, and other analogs show partial reversal by coenzyme Q6. Electron transport reactions inhibited are NADH and lactate oxidase, NADH menadione reductase, lactate phenazinemethosulfate reductase and duroquinol oxidase. The concentrations of DBMIB required are similar for electron transport and ATPase inhibition and inhibitions are all increased by uncouplers. Electron transport and ATPase are not inhibited in a DBMIB insensitive mutant. Soluble ATPase extracted from the membranes does not show DBMIB inhibition under either high or low Mg++ conditions. Lipophilic chelators show additional inhibition over DBMIB. It appears that coenzyme Q functions at three sites in E. coli electron transport where ATPase activity is controlled. Coenzyme Q deficient mutants also show decreased electron transport and ATPase activity which is restored by coenzyme Q.  相似文献   

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