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1.
Purification and characterization of a Ca2+/calmodulin-dependent protein kinase from rat brain 总被引:11,自引:0,他引:11
A soluble Ca2+/calmodulin-dependent protein kinase has been purified from rat brain to near homogeneity by using casein as substrate. The enzyme was purified by using hydroxylapatite adsorption chromatography, phosphocellulose ion-exchange chromatography, Sepharose 6B gel filtration, affinity chromatography using calmodulin-Sepharose 4B, and ammonium sulfate precipitation. On sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gels, the purified enzyme consists of three protein bands: a single polypeptide of 51 000 daltons and a doublet of 60 000 daltons. Measurements of the Stokes radius by gel filtration (81.3 +/- 3.7 A) and the sedimentation coefficient by sucrose density sedimentation (13.7 +/- 0.7 S) were used to calculate a native molecular mass of 460 000 +/- 29 000 daltons. The kinase autophosphorylated both the 51 000-dalton polypeptide and the 60 000-dalton doublet, resulting in a decreased mobility in NaDodSO4 gels. Comparison of the phosphopeptides produced by partial proteolysis of autophosphorylated enzyme reveals substantial similarities between subunits. These patterns, however, suggest that the 51 000-dalton subunit is not a proteolytic fragment of the 60 000-dalton doublet. Purified Ca2+/calmodulin-dependent casein kinase activity was dependent upon Ca2+, calmodulin, and ATP X Mg2+ or ATP X Mn2+ when measured under saturating casein concentrations. Co2+, Mn2+, and La3+ could substitute for Ca2+ in the presence of Mg2+ and saturating calmodulin concentrations. In addition to casein, the purified enzyme displayed a broad substrate specificity which suggests that it may be a "general" protein kinase with the potential for mediating numerous processes in brain and possibly other tissues. 相似文献
2.
Citrate lyase (EC 4.1.3.6) has been purified from Escherichia coli and the homogeneity of the preparation established from the three-component subunits obtained on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The purified enzyme has a specific activity of 120 mumol min-1 mg-1 and requires optimally 10 mM Mg2+ and a pH of 8.0 for the cleavage reaction. The native enzyme is polydispersed in the ultracentrifuge and in polyacrylamide gel electrophoresis. The enzyme complex is composed of three different polypeptide chains of 85 000, 54 000, 32 000 daltons. An estimate of subunit stoichiometry indicates that 1 mol of the largest polypeptide chain is associated with 6 mol each of the smaller ones. The polypeptide subunits have been isolated in pure state and their biological functions characterize. The 54 000-dalton subunit functions as the acyltransferase alpha subunit catalyzing the formation of citryl coenzyme A from citrate in the presence of acetyl coenzyme A and ethylenediaminetetraacetic acid. The 32 000-dalton subunit functions as the acyllyase beta subunit catalyzing the cleavage of (3S)-citryl coenzyme A to oxal-acetate and acetyl coenzyme A. The 85 000-dalton subunit, which carries exclusively the prosthetic group components, functions as the acyl-carrier protein gamma subunit in the cleavage of citrate in the presence of mg2+ and the alpha and beta subunits. The presence of a large ACP subunit and the unusual stoichiometry of the different subunits distinguish the complex from other citrate lyases. A ligase which acetylates the deacetyl[citrate lyase] in the presence of acetate and ATP has ben shown to be present in the organism.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
3.
The oligomycin- and N,N'-dicyclohexylcarbodiimide-sensitive adenosine triphosphatase complex extracted with Triton X-100 from the chromatophores of Rhodospirillum rubrum was extensively purified. The purification procedure included (diethylamino)ethylcellulose chromatography and glycerol gradient centrifugation. The specific activity of Mg2+-dependent ATP hydrolysis in the purified preparation increased about 11-fold, while that of Ca2+-dependent ATP hydrolysis increased 50-fold as compared with chromatophores. The purified adenosine triphosphatase complex dissociated into a maximum of eight different polypeptides upon electrophoresis in the presence of sodium dodecyl sulfate. The estimated subunit molecular weights were as follows: 56 000 (alpha), 50 000 (beta), 33 000 (gamma), and those ranging from 17 000 to 9400 for the remaining smaller subunits. The purified preparation was incorporated into phospholipid vesicles by using the freeze--thaw technique. The reconstituted vesicles catalyzed [32P]ATP exchange, which was almost completely inhibited by both oligomycin and N,N'-dicyclohexylcarbodiimide as well as by a protonophorous uncoupler, carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone. 相似文献
4.
Energy-linked reactions catalyzed by the purified ATPase complex (F0F1) from Rhodospirillum rubrum chromatophores 总被引:1,自引:0,他引:1
1. The isolation of F0F1-ATPase complex from Rhodospirillum rubrum chromatophores by the use of taurodeoxycholate is described. 2. The enzyme preparation contains about 12 polypeptides; five are subunits of the F1 moiety. 3. The ATPase activity of the purified enzyme is dependent on the addition of phospholipids. 4. Km-vales for Mg2+-ATP and Ca2+-ATP are similar to the values obtained for the membrane-bound enzyme. 5. The F0F1-ATPase complex is more than 70% inhibited by oligomycin and N,N'-dicyclohexylcarbodiimide. 6. The F0F1-ATPase complex was integrated into liposomes. The reconstituted proteoliposomes catalyzed energy transduction as shown by ATP-dependent quenching of acridine dye fluorescence and ATP-32Pi exchange. 相似文献
5.
The energy-linked ATPase complex has been isolated from spinach chloroplasts. This protein complex contained all the subunits of the chloroplast coupling factor (CF1) as well as several hydrophobic components. When the activated complex was reconstituted with added soybean phospholipids, it catalyzed the exchange of radioactive inorganic phosphate with ATP. Sonication of the complex into proteoliposomes together with bacteriorhodopsin yielded vesicles that catalyzed light-dependent ATP formation. Both the 32Pi-ATP exchange reactions and ATP formation were sensitive to uncouplers such as 3-tert-butyl-5,2′-dichloro-4′-nitrosalicylanilide, bis-(hexafluoroacetonyl)acetone and carbonyl cyanide-p-trifluoromethoxyphenyl-hydrazone, that act to dissipate a proton gradient. The energy transfer inhibitors dicyclohexylcarbodiimide, triphenyltin chloride and 2-β-d-glucopyranosyl-4,6′-dihydroxydihydrochalcone were also effective inhibitors of both reactions. 相似文献
6.
Ghosts derived from bovine chromaffin granules have a 32Pi-ATP exchange activity which is associated with the H+ pump of that membrane. This activity was low when compared to bacteria, chloroplasts or submitochondrial particles, but had similar properties (Km for ATP and Pi, ATP/Mg2+ ratio, pH profile, inhibition by dicyclohexylcarbodiimide and tributyltin) to the ATPase from above membranes. The 32Pi-ATP exchange activity was solubilized by cholate/octylglucoside mixtures. The soluble extract was lipid depleted by ammonium sulfate fractionation and partially purified by sucrose gradient centrifugation. The purified preparation was reconstituted with phospholipids by freeze-thawing. The reconstituted vesicles had a 32Pi-ATP exchange sensitive to dicyclohexylcarbodiimide and trybutyltin and an ATPase with a sensitivity to the inhibitors which varied with the reconstitution conditions. The α- and β-subunits of F1-ATPase were major components of the preparation. 相似文献
7.
The resolution-reconstitution approach has been employed in order to gain information as to the subunit composition of the Neurospora plasma membrane H+-ATPase. Proteoliposomes prepared from sonicated asolectin and a highly purified, radiolabeled preparation of the 105,000-dalton hydrolytic moiety of the H+-ATPase by a freeze-thaw procedure catalyze ATP hydrolysis-dependent proton translocation as indicated by the extensive 9-amino-6-chloro-2-methoxyacridine fluorescence quenching that occurs upon the addition of MgATP to the proteoliposomes, and the reversal of this quenching induced by the H+-ATPase inhibitor, vanadate, and the proton conductors, carbonyl cyanide m-chlorophenylhydrazone and nigericin plus K+. ATP hydrolysis is tightly coupled to proton translocation into the liposomes as indicated by the marked stimulation of ATP hydrolysis by carbonyl cyanide m-chlorophenylhydrazone and nigericin plus K+. The maximum stimulation of ATPase activity by proton conductors is about 3-fold, which indicates that at least two-thirds of the hydrolytically active ATPase molecules present in the reconstituted preparation are capable of translocating protons into the liposomes. Furthermore, as estimated by the extent of protection of the reconstituted 105,000-dalton hydrolytic moiety against tryptic degradation by vanadate in the presence of Mg2+ and ATP, the fraction of the total population of ATPase molecules that are hydrolytically active is at least 91%. Taken together, these data indicate that at least 61% of the ATPase molecules present in the reconstituted preparation are able to catalyze proton translocation. This information allows an estimation of the amount of any polypeptide in the preparation that must be present in order for that polypeptide to qualify as a subunit that is required for proton translocation in addition to the 105,000-dalton hydrolytic moiety, and an analysis of the radiolabeled ATPase preparation by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and urea rules out the involvement of any such polypeptides larger than 2,500 daltons. This indicates that the Neurospora plasma membrane H+-ATPase has no subunits even vaguely resembling any that have been found to be associated with other transport ATPases and that if this enzyme has any subunits at all other than the 105,000-dalton hydrolytic moiety, they must be very small. 相似文献
8.
The effect of trypsin on gastric (H+ + K+)-ATPase and K+-phosphatase was studied. Loss of both enzymic activities was biphasic, consisting of a fast and slow phase. Several peptides were produced from the original 105,000-dalton region of the sodium dodecyl sulfate electrophoretic separation, but only two, 87,000 and 47,000 daltons, were labeled following incubation with [gamma-33P]ATP. After a 30-min hydrolysis, 35% of the original peptide remained unaltered and appeared to be a glycoprotein. ATP and ADP abolished the second phase of tryptic inactivation of both activities and only two peptides, of 78,000 and 30,000 daltons, were found on the acrylamide gel in addition to the original 105,000-dalton region, neither of which was labeled by [gamma-33P]ATP. The protection was specific for these nucleotides, AMP, beta, gamma-methylene ATP, TTP, and pNPP being ineffective. Na+ and K+ at high concentrations reduced the rate of loss of activity but no change in the peptides produced was found. The level of phosphoenzyme was increased 2-fold by trypsin treatment, whereas the quantity of K+-sensitive phosphoenzyme remained relatively constant. Thus, the 105,000-dalton region is heterogeneous, consisting of a catalytic subunit (the active site is on a 47,000-dalton fragment), a glycoprotein, and another 105,000-dalton peptide. The action of trypsin is initially to prevent interconversion of a K+-insensitive to a K+-sensitive form of the phosphoenzyme, thus inhibiting hydrolysis. 相似文献
9.
Cold inactivation of vacuolar proton-ATPases 总被引:13,自引:0,他引:13
Incubation of the reconstituted H+-ATPase from chromaffin granules on ice resulted in inactivation of the proton-pumping and ATPase activities of the enzyme. Inactivation was dependent on the presence of Mg2+, Cl-, and ATP during the incubation at low temperature. Approximately 1 mM ATP, 1 mM Mg2+, and 200 mM Cl- were required for maximum inactivation. Incubation for about 10 min on ice was required to achieve 50% inactivation. A much smaller decline in activity was observed when the enzyme was incubated at room temperature with the same chemicals. Inactivation in the cold resulted in the release of five polypeptides from the membrane with apparent molecular masses of 72, 57, 41, 34, and 33 kDa on sodium dodecyl sulfate gels. Three of the polypeptides of 72, 57, and 34 kDa were identified as subunits of vacuolar H+-ATPases by antibody cross-reactivity. Similar results were obtained with several other vacuolar H+-ATPases including those from plant sources. It was concluded that the catalytic sector of the enzyme is released from the H+-ATPase complex by cold treatment, resulting in inactivation of the enzyme. 相似文献
10.
In the water-soluble fraction of rabbit skeletal muscle, at least two types of inorganic pyro phosphatase (PPase) are distinguishable on ion exchange column chromatography. One of them, pyrophosphatase-A (PPase-A), was isolated in an electrophoretically homogeneous form. This enzyme catalyzed the hydrolysis of PPi but not that of other phosphate esters. Only Mg2+ was required for activity and stability. Other cations such as Ca2+, Co2+, Mn2+, and Zn2+ had no activating effect. The activity of this PPase was optimum at pH 7.4. ATP, ADP, sodium imidodiphosphate (PNP), p-chloromercuribenzoate, and Ca2+ inhibited its enzymic activity. The enzyme was protected by dithiothreitol (DTT) against heat denaturation. The molecular weight was estimated to be 67,000 by gel filtration and the molecular size of the subunit was found to be 35,000 by gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). The enzyme probably consists of two identical subunits of 35,000 daltons. 相似文献
11.
Detergent (Lubrol WX)-solubilized sodium-potassium-activated adenosine triphosphatase ((Na+ + K+)-ATPase) of electrophorus electric organ contains two major constituent polypeptides with molecular weights of 96,000 and 58,000 which can be readily demonstrated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. These two polypeptides can be clearly separated and can be obtained in milligram quantities by preparative sodium dodecyl sulfate gel electrophoresis. The separated polypeptides, after removal of sodium dodecyl sulfate, and Lubrol-solubilized (Na+ + K+)-ATPase activity to some degree. Moreover, the degree of inhibition is directly proportional to the increasing amounts of antisera. The inhibition is maximal 4 weeks after the first injection. Immunodiffusion in 1% agar gel indicated that only Lubrol-solubilized enzyme antiserum, but not 58,000-dalton or 96,00-dalton polypeptide antiserum, gives one major precipitin band. However, specific complex formation between each polypeptide antiserum and Lubrol-solubilized enzyme occurs. This was demonstrated indirectly. After incubating Lubrol-solubilized enzyme with increasing amounts of polypeptide antisera at 37 degrees for 15 min, they were placed in the side wells of an immunodiffusion plate with antiserum against Lubrol-solubilized enzyme in the central well. The intensity of the precipitin band decreased with increasing amounts of polypeptide antisera. Thus, the results indicate that both 96,000-dalton and 58,000-dalton polypeptides are integral subunits of (Na+ + K+)-ATPase. 相似文献
12.
H Wohlrab 《Biochemistry》1979,18(10):2098-2102
The mitochondrial phosphate carrier is inhibited by the SH reagents p-(hydroxymercuri)benzoate and N-ethylmaleimide. Based on an analysis utilizing dodecyl sulfate-polyacrylamide gels, an SH-containing 32 000-dalton protein has been identified as a component of the phosphate carrier system. Two other N-[3H]ethylmaleimide-labeled proteins of the inner mitochondrial membrane have been eliminated from this role [Wholrab, H., & Greaney, J., Jr. (1978) Biochim. Biophys. Acta 503, 425] on the basis that band IV (45,000 daltons) is absent from heart sonic submitochondrial particles and band VII (6 500 daltons) does not react with p-(hydroxymercuri)benzoate. The mobility of the 32 000-dalton protein (0.43) is lower than that of the gamma subunit of the mitochondrial ATPase (0.46) and the carboxyatractyloside binding protein (0.48) on 12.5% dodecyl sulfate-polyacrylamide gels. In these flight muscle mitochondria, 0.87 nmol of N-[3H]ethylmaleimide per nmol of cytochrome a is bound to the 32,000-dalton protein. 相似文献
13.
Herpes simplex virus phosphoproteins. II. Characterization of the virion protein kinase and of the polypeptides phosphorylated in the virion. 总被引:34,自引:29,他引:5
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The protein kinase associated with purified herpes simplex virus 1 and 2 virions partitioned with the capsid-tegument structures and was not solubilized by non-ionic detergents and low, non-inhibitory concentrations of urea. The enzyme required Mg2+ or Mn2+ and utilized ATP or GTP. The activity was enhanced by non-ionic detergents and by Na+ even in the presence of high concentrations of of Mg2+, but not by cyclic nucleotides. The enzyme associated with capsid-tegument structures phosphorylated virion polypeptides only; exogenously added substrates (acidic and basic histones, casein, phosphovitin, protamine, and bovine serum albumin) were not phosphorylated. The major phosphorylated species were virion polypeptides (VP) 1-2, 4, 11-12, 13-14, 18.7, 18.8 and 23. VP 18.7 and VP 18.8 have not been previously detected, but may be phosphorylated forms of polypeptides co-migrating with VP 19. Of the remainder, only VP 23 has been previously identified as a capsid protein; the others are constituents of the tegument or of the under surface of the virion envelope. The distribution of the phosphate bound to viral polypeptides varied depending on the Mg2+ concentration and pH. In the absence of dithiothreitol, in vitro phosphate exchange was demonstrable in VP 23 and to a lesser extent in two other polypeptides on sequential phosphorylation frist with saturating amounts off unlabeled ATP and then with [gamma-32P]ATP. Analysis of the virion polypeptides specified by herpes simplex virus 1 X herpes simplex virus 2 recombinants indicates that the genes specifying the polypeptides which serve as a substrate for the protein kinase map in the unique sequences near the left and right reinterated DNA sequences of the L component. 相似文献
14.
Freshly purified preparations of anthranilate synthetase complex from Neurospora crassa appeared to be homogeneous on polyacrylamide disc gels and were composed of two distinct subunits, 94,000 and 70,000 daltons, respectively, as determined by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate. Carboxymethylation of the complex or treatment with guanidine hydrochloride and urea before sodium dodecyl sulfate treatment did not alter the subunit pattern. When the purified complex was iodinated with 125I- or methylated with [14C]dimethylsulfate, no labeled components other than the two subunits stained with Coomassie blue were detected after electrophoresis in the presence of sodium dodecyl sulfate. Although some purified preparations were stable, most were unstable upon storage. Analysis of the unstable preparations on nondenaturing and sodium dodecyl sulfate polyacrylamide disc gels revealed that the complex in these preparations was progressively fragmented to smaller components and subunits upon repeated freeze-thaw treatment or prolonged incubation at or above 4 degrees. Distinct fragments were generated ranging in size down to 25,000 daltons, and some fragments retained some of the activities associated with the anthranilate synthetase complex. On the basis of these and earlier studies, we conclude that anthranilate synthetase from Neurospora crassa is composed of two distinct subunits in an alpha2beta2 structure; one subunit is a trifunctional peptide which contains the catalytic sites for the phosphoribosylanthranilate isomerase and indoleglycerol phosphate synthetase reactions, and associates with the second subunit to form glutamine-dependent anthranilate synthetase. The smaller subunits and components previously reported for this complex are apparently due to protease activity present in purified preparations. 相似文献
15.
Reconstitution of the H+-ATPase complex of Rhodospirillum rubrum by the beta subunit of the chloroplast coupling factor 1 总被引:2,自引:0,他引:2
M L Richter Z Gromet-Elhanan R E McCarty 《The Journal of biological chemistry》1986,261(26):12109-12113
A method is described for isolating the beta subunit from spinach chloroplast F1 (CF1). The isolated beta subunit reconstituted an active F1 hybrid with the F1 of Rhodospirillum rubrum chromatophores from which the beta subunit had been removed. The CF1 beta subunit was similar to the isolated beta subunit of Escherichia coli F1 (Gromet-Elhanan, Z., Khananshivili, D., Weiss, S., Kanazawa, H., and Futai, M. (1985) J. Biol. Chem. 260, 12635-12640) in that it restored a substantial rate of ATP hydrolysis and low, but significant light-dependent ATP synthesis to the beta-less chromatophores. The low rate of photophosphorylation observed with the hybrid enzyme probably resulted from a looser coupling of the CF1 beta subunit to proton translocation in the R. rubrum Fo-F1 complex. The hybrid enzyme exhibited a high specificity for Mg2+-ATP as substrate for ATP hydrolysis and both ATP synthesis and hydrolysis were strongly inhibited by the antibiotic tentoxin. In contrast, chromatophores reconstituted with the native R. rubrum beta subunit actively hydrolyzed both Mg2+-ATP and Ca2+-ATP and were insensitive to tentoxin. These results indicate a close functional homology between the beta subunits of the prokaryotic and eukaryotic H+-ATPases and suggest a role for the beta subunit in conferring the different metal ion specificities and inhibitor sensitivities upon the enzymes. They also demonstrate the feasibility of isolating the beta subunit from CF1 in a reconstitutively active form. 相似文献
16.
Purification and characterization of the F1-ATPase from Clostridium thermoaceticum. 总被引:6,自引:6,他引:0
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The F1 portion of the H+-ATPase from Clostridium thermoaceticum was purified to homogeneity by solubilization at low ionic strength, ion-exchange chromatography, and gel filtration. The last indicated the Mr to be 370,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the pure enzyme revealed four bands with Mr corresponding to 60,000, 55,000, 37,000, and 17,000 in an apparent molar ratio of 3:3:1:1. The purified enzyme would bind to stripped membranes to reconstitute dicyclohexylcarbodiimide-sensitive ATPase activity. Phosphohydrolase activity, measured at 58 degrees C, was optimal at pH 8.5. In the presence of a 1 mM excess of Mg2+ over the concentration of ATP, the Km for ATP was 0.4 mM, and the Vmax was 6.7 mumol min-1 mg-1. Unlike the membrane-bound F1F0 complex, the F1-ATPase was relatively insensitive to the inhibitors dicyclohexylcarbodiimide and tributyltin chloride. Both the complex and the F1-ATPase were inhibited by quercetin, azide, 7-chloro-4-nitro-benz-2-oxa-1,3-diazole, and free magnesium, and both were stimulated by primary alcohols and sulfite. In whole cells, the F1F0-ATPase catalyzed the synthesis of ATP in response to a pH gradient. 相似文献
17.
Target molecular weight of the gastric (H+ + K+)-ATPase functional and structural molecular size 总被引:5,自引:0,他引:5
G Saccomani G Sachs J Cuppoletti C Y Jung 《The Journal of biological chemistry》1981,256(15):7727-7729
The state of assembly of the (H+ + K+)-ATPase in purified hog gastric mucosa membranes was studied by target size analysis applied to radiation-induced enzyme inactivation and polypeptide degradation data. Radiation inactivated the Mg2+-ATPase, K+-stimulated ATPase, and p-nitrophenyl phosphatase activities of the membrane preparation with a dose dependence characteristic of a target size of 270,000-daltons. Radiation also bleached the major 100,000-dalton sodium dodecyl sulfate-gel electrophoresis band of this preparation, indicating a radiation-induced degradation. This apparent polypeptide degradation exhibited a dose dependency corresponding to a target size of 250,000 daltons in situ. It is suggested that the gastric ATPase is a trimeric assembly of the 100,000-dalton polypeptides. 相似文献
18.
Calmodulin-mediated regulation of calcium transport and (Ca2+ + Mg2+)-activated ATPase activity in isolated cardiac sarcoplasmic reticulum 总被引:6,自引:0,他引:6
A severalfold activation of calcium transport and (Ca2+ + Mg2+)-activated ATPase activity by micromolar concentrations of calmodulin was observed in sarcoplasmic reticulum vesicles obtained from canine ventricles. This activation was seen in the presence of 120 mM KCl. The ratio of moles of calcium transported per mol of ATP hydrolyzed remained at about 0.75 when calcium transport and (Ca2+ + Mg2+)-activated ATPase activity were measured in the presence and absence of calmodulin. Thus, the efficiency of the calcium transport process did not change. Stimulation of calcium transport by calmodulin involves the phosphorylation of one or more proteins. The major 32P-labeled protein, as determined by sodium dodecyl sulfate slab gel electrophoresis, was the 22,000-dalton protein called phospholamban. The Ca2+ concentration dependency of calmodulin-stimulated microsomal phosphorylation corresponded to that of calmodulin-stimulated (Ca2+ + Mg2+)-activated ATPase activity. Proteins of 11,000 and 6,000 daltons and other proteins were labeled to a lesser extent. A similar phosphorylation pattern was obtained when microsomes were incubated with cAMP-dependent protein kinase and ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. Phosphorylation produced by added cAMP-dependent protein kinase and calmodulin was additive. These studies provided further evidence for Ca2+-dependent regulation of calcium transport by calmodulin in sarcoplasmic reticulum that could play a role in the beat-to-beat regulation of cardiac relaxation in the intact heart. 相似文献
19.
The cytoplasmic membrane of micoplasmic cells, in particular of A. laidlawii cells, contains a proton-carrier Mg2+ -activated ATPase. A whole H+ -ATPase complex (F0-F1) was isolated from these cells and characterized. The isolation procedure included solubilization of the enzyme with Triton X-100 followed by ion-exchange chromatography on DEAE-cellulose and gel filtration on Sepharose 6B. The enzyme was inhibited by dicyclohexylcarbodiimide (10(-4) M). The Km value for ATP hydrolysis and Ki for ADP hydrolysis were determined. The order of the constants did not differ from those measured earlier for factor F1 of the complex. The purified enzyme, similar to its hydrophylic moiety is sensitive to the action of bivalent cations. The subunit composition of the whole complex and of its water-soluble part was investigated. The complex was found to contain 11 polypeptides, five of which belong to factor F1. The molecular weights of these polypeptides were determined. 相似文献
20.
R Kr?mer 《Biochimica et biophysica acta》1980,592(3):615-620
1. Divalent cations cause a decrease in the exchange activity of the reconstituted ADP,ATP translocator from beef heart mitochondria. This effect is due to complex formation with the adenine nucleotides. 2. It is confirmed that only the free nucleotides are transported. A possible competition of free adenine nucleotides and the Mg2+-complexes for the binding site at the carrier protein is excluded. 3. The stability constants (Kn) for the cation-nucleotide complexes are derived from these experiments. For Mg2+-ATP, Kn = 0.8 x 10(4) M-1 and for Mg2+-ADP, Kn = 0.8 x 10(3) M-1 is obtained. 4. The carrier system was reconstituted with the neutral phospholipids phosphatidylcholine and phosphatidylethanolamine. Interaction of the divalent cations with these phospholipids seem not to be important for the exchange suppression. 相似文献