首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 15 毫秒
1.
The three major subunits (α, β and γ) of the coupling factor, F1 ATPase, of Escherichia coli were separated and purified by hydrophobic column chromatography after the enzyme was dissociated by cold inactivation. The ability to hydrolyze ATP was reconstituted by dialyzing the mixture of subunits against 0.05 M Tris-succinate, pH 6.0, containing 2 mM ATP and 2 mM MgCl2. A mixture containing α, β and γ regained ATP hydrolyzing activity. Individual subunits alone or mixtures of any two subunits did not develop ATPase activity, except for a low but significant activity with α plus β. The reconstituted ATPase had a Km of 0.23 mM for ATP and a molecular weight by sucrose gradient density centrifugation of about 280,000.  相似文献   

2.
The phospholipid requirement of the (Ca2+ + Mg2+)-ATPase present in a membrane fraction from human platelets was studied using various purified phospholipases. Only those phospholipases, which hydrolyse the negatively charged phospholipids, inhibited the (Ca2+ + Mg2+)-ATPase activity. The ATPase activity could be restored by adding mixed micelles of Triton X-100 and phosphatidylserine or phosphatidylinositol. Micelles with phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine or sphingomyelin could not be used for reconstitution and inhibited the activity of the native enzyme.  相似文献   

3.
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   

4.
The ATPase activity of rabbit-kidney brush border can be activated almost equally well by Ca2+ and Mg2+ and, therefore, should be called (Ca2+ or Mg2+)-ATPase. This enzyme was solubilized and enriched 14-fold by the following steps: pretreatment with papain removed 69% of alkaline phosphatase without attacking a significant portion of the ATPase activity. Addition of 1% cholate removed 65% of the protein but no ATPase activity. The combination of cholate (0.5%) and deoxycholate (0.4%) solubilized most of the ATPase activity and most of the remaining protein. A column chromatography of the extract on Sepharose CL-2B resulted in an 6.5-fold increase of specific ATPase activity. A precipitation by ammonium sulfate (40% saturation) produced an additional 1.9-fold increase. The yield of this partial purification was 16%. Towards the nucleotides UTP and GTP the enzyme showed an activity slightly higher, and towards ITP and CTP an activity slightly lower than that with ATP. ADP was split about half as fast as ATP. AMP was not accepted by the enzyme. Replacing MgCl2 by CaCl2 resulted in an ATPase activity of 92% of that with MgCl2. Using calcium- and magnesium-ATP as substrates, apparent Km values of 0.22 and 0.33 mM, respectively, were obtained. The gel electrophoresis revealed the enrichment of a protein with an apparent Mr of 95 000 and also that of microvillus actin.  相似文献   

5.
Limited chymotryptic cleavage of the α subunits in the solubilized ATPase from Streptococcus faecalis is accompanied by loss of membrane binding capacity (Abrams, A., Morris, D., Jensen, C. (1976) Biochem. 15, 5560). To obtain evidence that the α chains might function directly in membrane attachment we compared the effect of chymotrypsin on the soluble and membrane-bound enzyme. Using a low level of chymotrypsin the soluble ATPase was quantitatively converted to a catalytically active form in which the 55000 dalton α chains were shortened by approximately 2000 daltons. However, at 80 fold higher levels of chymotrypsin the ATPase in a reconstituted ATPase-membrane complex was completely unaffected. Protection from chymotryptic attack appeared to be membrane specific since the soluble ATPase was not protected by addition of massive amounts of bovine serum albumin. The total and specific immunity to chymotrypsin conferred by membrane binding indicates that chymotrypsin-sensitive α chain “tails” are closely associated with or buried in the membrane. These findings support the view that the α chains are involved directly in membrane attachment.  相似文献   

6.
Rat testis mitochondrial ATPase was not inhibited by oligomycin at pH 7.5. It was inhibited only at higher alkaline pH's, and showed a lower sensitivity both to oligomycin and N,N′-dicyclohexylcarbodiimide and a higher one to efrapeptin. In submitochondrial particles, testis ATPase was only slightly inhibited by oligomycin, ossamycin, and efrapeptin. The possibility of a loose binding of F1 to the membrane was supported by its recovery from the supernatant of the submitochondrial particles. Furthermore, by electron microscopy, after hypoosmotic shock and negative staining of the mitochondrial preparations, most of the inner mitochondrial membranes showed only a few “knobs” or none at all. The capacity of the testis mitochondrial preparation to produce ATP was tested and compared to that from liver. ATP synthetase/ATPase activity ratio was 301 in liver mitochondria, whereas in the testis it was 31. In spite of this large difference, at least part of the testis ATPase must be firmly bound to the membrane, since it is able to form ATP. The rest seems to be loosely bound and its functional significance is still unknown.  相似文献   

7.
Cholinergic synaptic vesicles from the electric organ of Torpedocalifornica have been subjected to analytical scale separation techniques not utilized in the isolation procedure, and the ATPase activity of separated fractions determined. Most of the ATPase activity migrated with the vesicles. Sensitivity of the ATPase activity to 16 potential inhibitors also was determined. Most of the ATPase activity was inhibited by low concentrations of 4-chloro-7-nitrobenzo-oxadiazole (NBD-C1) and dicyclohexylcarbodiimide (DCCD), but not by a water soluble carbodiimide. The close association of the ATPase with the vesicles and the pattern of inhibition obtained provide further support for the authentic presence of a membrane bound Ca2+Mg2+ ATPase in the cholinergic synaptic vesicle.  相似文献   

8.
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   

9.
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   

10.
Removal of the F1 ATPase from membrane vesicles of Escherichiacoli resulted in leakage of protons across the membrane through the FO portion of the ATPase complex. The leakage of protons was prevented by antiserum to the N,N′-dicyclohexylcarbodiimide (DCCD)-binding polypeptide in everted but not in “right-side out” membrane vesicles. The antiserum prevented the rebinding of F1 ATPase to F1-stripped everted membrane vesicles. It is concluded that in F1-depleted vesicles the DCCD-binding polypeptide is exposed on the cytoplasmic surface of the cell membrane at or close to the binding site of the F1 ATPase.  相似文献   

11.
The surface activity and enzymic properties of the factor F1, the catalytic moiety of Streptococcus faecalis H+-ATPase, has been studied at the air-water and phospholipid-water interfaces. F1 does not interact with the monolayer phospholipids, hence its adsorption on a biological membrane must be due mainly to its recognition of proteins of the hydrophobic complex. The dimensions of the F1 molecule at the air-water interface have been estimated. In the presence of Mg2+, base area is S = 1.8 · 104A?2, height h = 27 A?. Bearing in mind the size of a globular subunit, it follows from the measurements that the major F1 subunits should all lie in the same plane. The ATPase activity of F1 at the interface is inversely proportional to the monolayer density. With low density monolayer, the specific ATPase activity is higher at the interface than in the bulk of the solution.Adsorption of F1 at the interface shifts the isoelectric point of the protein, apparently due to changes in its conformation. The findings are discussed relative to the proton-active transport mechanism.  相似文献   

12.
NMR studies in D2O (>90%) reveal that Alanine Racemase (5.1.1.1.) from B. subtilis catalyzes the exchange of the α hydrogen of D- and L-alanine with D2O. Glutamic Pyruvic Transaminase (2.6.1.2.) and Glutamic Oxaloacetic Transaminase (2.6.1.1.) catalyze the exchange of α and β hydrogens of L-alanine. The rates of exchange of α and β hydrogens appear to be of the same order of magnitude. The transaminase catalyzed exchange is enhanced by catalytic amounts of pyruvate. The side chain of L-alanine is held more rigidly at the active site of transaminase so that the planar conjugated system can be extended to include the α and β carbons. A generalized mechanism is proposed for the action of pyridoxal phosphate dependent transaminases which extends Braunstein and Snell mechanism to include the structures which contribute to the labilization of β hydrogens of amino acids by the transaminases that have been studied.  相似文献   

13.
The sarcolemmal membrane obtained from rat heart by hypotonic shock-LiBr treatment method was found to incorporate 32P from [γ-32P] ATP in the absence and presence of cyclic AMP and protein kinase. The phosphorylated membrane showed an increase in Ca2+ ATPase and Mg2+ ATPase activities without any changes in Na+K+ ATPase activity. The observed increase in Ca2+Mg2+ ATPase activity was found to be associated with an increase in Vmax value of the reaction whereas Ka value for Ca2+Mg2+ was not altered. These results provide information concerning biochemical mechanism for increased calcium entry due to hormones which are known to elevate cyclic AMP levels in myocardium and produce a positive inotropic effect.  相似文献   

14.
A study has been made to determine whether renal plasma membranes contain an HCO3? stimulated, ouabain insensitive Mg ATPase. Purified mitochondrial, microsomal and brush border membrane fractions have been isolated from rabbit kidney.The microsomal anion-sensitive ATPase activity appears to be entirely of mitochondrial origin on the basis of the effects of inhibitors of mitochondrial Mg ATPase.The brush border membrane fraction is contaminated with mitochondrial fragments and contains an Mg ATPase activity with low anion-sensitivity. Further purification of this fraction causes parallel decreases in anion-sensitivity of the Mg ATPase activity and in cytochrome c oxidase activity.These results indicate that conclusions previously reached by other investigators for a role of anion-sensitive Mg ATPase in the bicarbonate reabsorption of the proximal tubule may no longer be tenable.  相似文献   

15.
ATPase activity of coupled Zajdela hepatoma mitochondria was rendered uncoupler-sensitive by decreasing free fatty acids content in mitochondria or by preincubation of mitochondria with ATP prior to the addition of an uncoupler. The latter treatment resulted in an accelerated transport of ATP into the organelles. The effect of carbonylcyanide-m-chlorophenylhydrazone and oligomycin on the decrease of the ATP content in whole Zajdela hepatoma cells indicated that the hepatoma mitochondrial ATPase is stimulated by uncouplers invivo. The conclusion is that the uncoupler-insensitive ATPase activity of coupled Zajdela hepatoma mitochondria is exhibited only by isolated organelles and results from a reduced ATPADP translocase activity.  相似文献   

16.
Photosynthetic membranes derived from sonic extracts of the cyanobacterium Spirulinaplatensis contain a latent Ca+2-ATPase which is activated by exposure to trypsin. When sonic membranes are washed with ethylenediaminetetraacetic acid, the ATPase is removed from these membranes with an accompanying loss of photophosphorylation activity. The latent ATPase activity solubilized by washing has been partially purified, and addition of the enzyme to depleted membranes restores photophosphorylation activity to levels approaching 50% of the rates observed in unwashed membranes. These data indicate that this ATPase is the coupling factor responsible for photosynthetic energy transduction in Spirulinaplatensis.  相似文献   

17.
Incubation of F1 in the presence of Mg2+ results in a pronounced lag in its ATPase activity measured with the ATP-regenerating system. A decrease of the initial rate of ATPase induced by Mg2+ is also observed when free nucleotides were separated from the enzyme by Sephadex gel filtration. No inhibition is observed when F1 treated to remove tightly bound nucleotides was preincubated in the presence of Mg2+. Mg2+-induced inhibition of ATPase activity of nucleotide-depleted F1 can be restored by an addition of low concentrations of ADP. In all cases the inhibited ATPase can be activated by the ADP-removing system /phosphoenol pyruvate + pyruvate kinase/. It is concluded that i/ Mg2+-induced inhibition of the ATPase activity of F1 is due to the formation of an inactive F1. ADP complex; and ii/ unusual inhibition of oligomycin-sensitive ATPase by ADP /Fitin et al., Biochem. Biophys. Res. Communs. 1979, 86, 434/ is directed to F1 component of the complete mitochondrial ATPase system.  相似文献   

18.
Short, mild treatments of sarcoplasmic reticulum vesicles with aqueous n-alcohols from methanol to n-heptanol caused an inhibition of calcium uptake and an enhancement of ATPase activity. The n-alcohol treatments increased both calcium-dependent (extra) ATPase activity and calcium-independent (basic) ATPase activity of vesicles. The apparent initial reaction rate of ATPase of n-alcohol-treated vesicles was about twice that of control vesicles. With increasing number (n) of carbon atoms of the n-alcohols, the maximum increment of ATPase activity increased, and both the alcohol concentration (NCa) required to inhibit calcium uptake by 50% and the alcohol concentration (NATPase) required to enhance ATPase activity by 50% of the maximum increment of ATPase activity decreased as follows.
NCa=23.5·10?0.593nM
NATPase=35.5·10?0.593nM
The ratio, NATPase to NCa, was constant for all n values. The apparent free energy of binding of the methylene groups of n-alcohols to sarcoplasmic reticulum vesicles was evaluated (?796 cal/mole) and compared with data from the partition of n-alcohols in octanol and water (?670 cal/mole). The effects of n-alcohols on membrane vesicles are discussed on the basis of these data.  相似文献   

19.
In intact soybean roots, chlorpromazine causes a depolarization of the membrane potential at low concentrations (as low as 30 μM, half-maximally at about 150 μM), and induces a marked decrease in ATP levels at higher concentrations (half-maximal at about 0.5 mM) over longer periods of time. In root microsomal suspensions, chlorpromazine inhibits an apparently specific ATPase activity component (half-maximally at about 0.3 mM). Chlorpromazine inhibits N,N′-dicyclohexylcarbodiimide-, diethylstilbesterol- and azide-inhibited ATPase activities. On linear sucrose gradients, chlorpromazine inhibition of ATPase activity occurs in two peaks, at 1.12 g/ml and 1.14–1.17 g/ml, which may represent a tonoplast and plasma membrane ATPase, respectively. Neither peak corresponds to the F1 ATPase. It is unclear whether ATPase inhibition or ATP loss is the cause of the membrane potential depolarization. Clearly chlorpromazine has multiple effects which are probably unrelated to its calmodulin-inhibition activity.  相似文献   

20.
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   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号