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1.
Brush border membrane vesicles (BBMV) from the midgut epithelial cells of silkworm larvae were prepared. ATP hydrolyzing activity (ATPase activity) was associated with the BBMV. ATPase activity without Mg2 + was not observed at pH 7 but substantial ATP hydrolyzing activity was observed at pH 7 with Mg2 +. The enzyme required Mn2 +, Mg2 +, or Ca2+ ions. The enzyme also hydrolyzed ITP and GTP but not p-NPP, ADP, or AMP. KNO3 and NEM strongly inhibited the ATPase activity. Behaviours of the ATPase against inhibitors suggested that it resembled vacuolar type ATPase.  相似文献   

2.
Summary The hydrolysis of ATP, AMP and glycerophosphate (GP) at alkaline pH in mineralizing bone and teeth of young mice has been studied histochemically. The substrates were visibly hydrolyzed to the same degree in osteoblasts, cells of stratum intermedium, odontoblasts and subodontoblasts at Ca2+ concentrations ranging from 10 mM to 600 mM. In the ameloblasts, however, only ATP was hydrolyzed. The ATPase activities gradually decreased at increasing Mg2+/Ca2+ ratios. The AMPase and GPase activities, on the other hand, were visibly unaffected. Marked cellular staining, including the nuclei was seen with AMP and GP as substrates when only Mg2+ ions were added. No ATPase activity at all could be recorded in media containing Mg2+ but no Ca2+ ions. The different phosphatase activities in cells involved in hard tissue formation were identically affected by preincubations with solutions containing various concentrations of Ca2+ or Mg2+ ions. The ATPase activity in striated muscle fibres and blood vessel walls, however, was affected differently by the same procedure.The results indicate that the phosphatase activities recorded in osteoblasts, cells of stratum intermedium, odontoblasts and subodontoblasts at alkaline pH belong to one single enzyme. The results also imply that CaATP is the preferred substrate in the enzymatic hydrolysis of ATP in hard-tissue-forming cells.  相似文献   

3.
Both oxalate-supported Ca2+ uptake and Ca2+-stimulated ATPase activity of the sarcoplasmic reticulum are sensitive to the pH of the assay medium. Ca2+ uptake is optimal at relatively acidic pH (6.2–6.6); whereas, Ca2+-stimulated ATPase activity is optimal at a more alkaline pH (7.4–8.0). Following the addition of ATP, Ca2+ uptake demonstrates a time-dependent resistance to the inhibition by an alkaline pH. Once the linear phase of Ca2+ uptake is reached, alkalinization thereafter does not alter the rate established at the acidic pH. A similar time-dependent resistance is observed to the inhibition of Ca2+ uptake by the cation ionophore, X537A. In contrast, acidification of the alkaline medium after Ca2+ uptake is initiated by ATP has no such resistance to change. Acidification results in a prompt acceleration of the rate of Ca2+ uptake identical to that observed under control conditions at the acidic pH. Ca2+-stimulated ATPase activity, however, increases with alkalinization and decreased with acidification, regardless of time, in a manner expected from the rates observed under conditions when the pH is constant from the time of ATP addition. The results suggest that there is a time-dependent, pH-sensitive factor of oxalate-supported Ca2+ uptake. This factor can be activated by acidification at any time after ATP addition and, thus, does not represent a destruction of membrane function. In contrast, Ca2+-stimulated ATPase activity demonstrates no time-dependent resistance to pH change.  相似文献   

4.
The turnip (Brassica rapa L.) microsome fraction contains both a Mg2+-inhibited acid phosphatase and a salt-stimulated Mg2+-activated ATPase. However, as the pH optimum of the ATPase was 8.0 to 8.5, the acid phosphatase activity could be eliminated by assaying at or above pH 7.8. The ATPase was concentrated in a fraction equivalent to the smooth microsomal membranes and was not due to fragments of mitochondria. The salt-stimulated activity showed specificity for anions rather than cations. The activity was further stimulated by carbonyl cyanide m-chloro-phenylhydrazone (CCCP), 2,4-dinitrophenol, valinomycin, nigericin, and NH4Cl. There was a synergistic effect between CCCP and valinomycin. Activity was insensitive to oligomycin phlorizin, ouabain, and atractylate. Based on similarity to the chloroplast ATPase, it was proposed that this ATPase was situated on the outside of the vesicle.  相似文献   

5.
Microsomal fractions from wheat (Triticum vulgare) and oat (Avena sativa) roots were used to study Mg2+ and Ca2+ activated adenosine triphosphatases, their dependence of pH, and how Mg2+ and Ca2+ compete or add in stimulation and inhibition. Wheat gives a high proportion of Ca2+ stimulated ATPase. Less effect is obtained with Mg2+. The characteristics of oar ATPase are the reverse. The ATPase from the wheat roots depends on the mineral nutrition. A kinetïc analysis shows one site, where Mg2+ and Ca2+ at low concentrations (or complexes between the di-valents and ATP) cooperate in the activation of the ATPase. The action of this site is more dearly expressed at pH 6.0 than at 6.8, and more clearly in the preparations from low salt roots than in those from high salt conditions. In another site, which is particularly evident in preparations from high salt roots tested at pH 6.8, high concentrations of Mg2+ inhibit the ATPase; this inhibition is competitively relieved by Ca2+. The specific activity of the ATPase from high salt roots of wheat is higher than that from low salt roots, although the amount of protein of the fraction studied remains the same, when calculated per g fresh weight of the roots.  相似文献   

6.
Summary Experiments are reported demonstrating that differential rates of inactivation of the histochemical staining for myofibrillar actomyosin ATPase in rat skeletal muscle fibers exist following inclusion of low concentrations of Cu2+ in the preincubation medium. This response of rat muscle occurs at near neutral (7.40), acid (4.60), and alkaline (10.30) pH. The response to Cu2+ appears to result from a binding of Cu2+ onto the myofibrillar complex, probably on myosin itself, as it can be reversed by soaking of the pretreated muscle sections in sodium cyanide or the Cu2+ chelator diethyldithiocarbamate. The pattern of modification of the staining pattern following pretreatment with Cu2+ is the mirror image of that produced by pretreatment with acid. The results demonstrate that the inclusion of Cu2+ in the preincubation media for the myofibrillar actomyosin ATPase can be a useful tool to differentiate fiber types. They also support the earlier conclusion that three distinct types of type II fibers can be identified in rat skeletal muscle based on the histochemical staining for myofibrillar actomyosin ATPase.  相似文献   

7.
《Experimental mycology》1987,11(1):49-59
Plasma membrane vesicles were isolated fromPhytophthora megasperma f. sp.glycinea using conventional methods of mechanical disruption followed by differential and density gradient centrifugation. The validity of presumed biochemical markers was confirmed using electron microscopy and the phosphotungstic acid-chromic acid staining procedure, which was judged to be specific for plasma membrane when performed under suitable conditions. The plasma membrane fraction showed a peak equilibrium density of 1.14 g/ml and was identified by its vanadate-sensitive Mg2+-dependent ATPase with an optimum temperature of 42°C and a pH optimum of 6.0 to 6.5. The activity was weakly stimulated by K+ and strongly inhibited by Ca2+. The enzyme showed a marked specificity for ATP as a substrate compared to other nucleoside mono-, di-, and triphosphate substrates or other general phosphatase substrates. The divalent cation requirement could be met equally well by Mg2+ and Co2+ and, to a lesser extent, by Mn2+, but not by Ni2+, Ba2+, Zn2+, Sr2+, Ca2+, Hg2+, Cu2+, or Fe2+ (in decreasing order of preference). Contamination by intact mitochondria (density 1.21 g/ml) or mitochondrial fragments (density 1.16 g/ml) was minimal and could be monitored by measuring cytochromec oxidase or oligomycin-sensitive pH 8.5 ATPase.  相似文献   

8.
The preparation and characterization of a Ca2+-sensitive actomyosin from chicken gizzard is described. The pH curve of the Mg2+ ATPase activity of the actomyosin was dominated by the activity of the myosin component, and this gave rise to the acid and alkaline optima. Skeletal muscle myosin showed a similar curve. Both the activation of myosin ATPase by actin, and the Ca2+ sensitivity were confined to the neutral pH region. The subunit composition of the Ca2+-sensitive actomyosin was interesting in that no components corresponding to skeletal muscle troponin were obvious. It is suggested that the activity of gizzard actomyosin is regulated by a protein on the thin filaments with a subunit weight of ~130,000.  相似文献   

9.
A simple method was developed for selective solubilization of membrane ATPase from etiolated corn seedlings using 0.01% Triton X100 and 0.01% deoxycholate containing 200 mM KI. An 81-fold enriched enzyme preparation, with specific activity of 133 μmol Pi/mg protein/hr, was obtained. The enzyme stored in 25 mM Tris-HCl buffer (pH 7.5) at 4° showed rapid loss of activity. The enzyme was stabilized by 1 mM EDTA with addition of 1.2 mM Mg2+°. Mg2+ and Ca2+ (1.2 mM) increased enzymatic activity by 12 and 10.8% respectively, whereas Na+ and K+ brought about a 20% increase in ATP-hydrolysis. The effect of combined mono- and di-valent ions was neither synergistic nor additive. Ouabain exerted no effect on enzyme activity. The enzyme showed two pH optima (6.0 and 7.5) in the presence of Na+ and K+, and one optimum at pH 6.5 in the absence of these ions. On polyacrylamide gel the enzyme was resolved into two protein bands, both exhibiting ATPase activity. It is suggested that the soluble enzyme from the microsomal fraction of corn seedlings contains two ATP-hydrolyzing enzymes, one of them being stimulated by Na+ and K+ ions.  相似文献   

10.
In order to gain some information regarding Ca2+-dependent ATPase, the enzyme was purified from cardiac sarcolemma and its properties were compared with Ca2+-ATPase activity of myosin purified from rat heart. Both Ca2+-dependent ATPase and myosin ATPase were stimulated by Ca2+ but the maximal activation of Ca2+-dependent ATPase required 4 mM Ca2+ whereas that of myosin ATPase required 10 mM Ca2+. These ATPases were also activated by other divalent cations in the order of Ca2+ > Mn2+ > Sr2+ > Br2+ > Mg2+; however, there was a marked difference in the pattern of their activation by these cations. Unlike the myosin ATPase, the ATP hydrolysis by Ca2+-dependent ATPase was not activated by actin. The pH optima of Ca2+-dependent ATPase and myosin ATPase were 9.5 and 6.5 respectively. Na+ markedly inhibited Ca2+-dependent ATPase but had no effect on the myosin ATPase activity. N-ethylmaleimide inhibited Ca2+-dependent ATPase more than myosin ATPase whereas the inhibitory effect of vanadate was more on myosin ATPase than Ca2+-dependent ATPase. Both Ca2+-dependent ATPase and myosin ATPase were stimulated by K-EDTA and NH4-EDTA. When myofibrils were treated with trypsin and passed through columns similar to those used for purifying Ca2+-ATPase from sarcolemma, an enzyme with ATPase activity was obtained. This myofibrillar ATPase was maximally activated at 3–4 mM Ca2+ and 3 to 4 mM ATP like sarcolemmal Ca2+-dependent ATPase. K+ stimulated both ATPase activities in the absence of Ca2+ and inhibited in the presence of Ca2+. Both enzymes were inhibited by Na+, Mg2+, La3+, and azide similarly. However, Ca2+ ATPase from myofibrils showed three peptide bands in SDS polyacrylamide gel electrophoresis whereas Ca2+ ATPase from sarcolemma contained only two bands. Sarcolemmal Ca2+-ATPase had two affinity sites for ATP (0.012 mM and 0.23 mM) while myofibrillar Ca2+-ATPase had only one affinity site (0.34 mM). Myofibrillar Ca2+-ATPase was more sensitive to maleic anhydride and iodoacetamide than sarcolemmal Ca2+-ATPase. These observations suggest that Ca2+-dependent ATPase may be a myosin like protein in the heart sarcolemma and is unlikely to be a tryptic fragment of myosin present in the myofibrils.  相似文献   

11.
The ability of CASF (Ca2+-activated sarcoplasmic factor), a proteolytic enzyme that has recently been isolated from muscle and that removes Z-disks from myofibrils, to remove soluble material from myofibrils and to alter the Mg2+-modified ATPase activity of myofibrils was studied. A new assay involving determination of soluble material released from myofibrils was developed to measure CASF activity quantitatively. Optimum pH and optimum Ca2+ concentration for CASF activity as determined by this new assay were 7.0 and 1 mm, respectively. Proteolytic activity of CASF on myofibrils was prevented completely by excess EDTA. CASF treatment of myofibrils at CASF to myofibril ratios of 1: 20 by weight for 30 min caused a 20~25% increase in Mg2+-modified ATPase activity. CASF treatment for 360 min under these same conditions caused a decrease in Mg2+-modified ATPase activity at the highest ionic strengths used in this study (46.7 and 66.7 mm KCI). The increase in Mg2+-modified ATPase activity may originate from CASF degradation of troponin, whereas the decrease in Mg2+- modified ATPase activity may be due to CASF destruction or release of α-actinin from myofibrils. Digestion of myofibrils by CASF causes in the myofibrils (degradation of Z-lines, increase of ATPase activity) that are very similar to the changes caused by postmortem storage.  相似文献   

12.
Summary Cytochemical investigations of ATPase activity were performed on lymphocytes isolated from peripheral blood and activatedin vitro by phytohaemagglutinin or by the two-way mixed lymphocyte reaction. Uncultured lymphocytes showed very little activity localized in small granules. The activity increased markedly during transformation. In fully transformed and actively proliferating cells, the ATPase activity was intense and localized in a crescentic perinuclear area of cytoplasm which was pale-staining and vesicular in Giemsa-stained preparations. In mitotic cells, the activity was in discrete granules or elongated structures suggestive of mitochondria, scattered throughout the cytoplasm. The ATPase activity had a pH optimum of 8.5 to 9.5 and was strongly inhibited at pH 7.5. The activity was stimulated by Ca2+ and Mg2+ and was inhibited byp-chloromercuribenzoate but not by oligomycin, which appeared to enhance the reaction. Lead nitrate at a concentration of 3mm did not inhibit the reaction.  相似文献   

13.
Summary The distribution of Mg+ +-ATPase in osteoclasts along the endosteal surface of the chick tibia was investigated by neutral and alkaline pH cytochemical methods at the electron-microscopic level. Reaction product was observed in mitochondria, cytoplasmic vesicles, and ruffled-border membrane. Levamisole, ouabain, and vanadate did not affect the enzymatic activity. Para-chloromercuribenzoic acid (PCMB) prevented staining of mitochondria, ruffled border, and most cytoplasmic vesicles. Tri-n-butyltin decreased the amount of reaction product in cytoplasmic vesicles and ruffled-border membrane, but did not inhibit reaction product formation within mitochondria. Duramycin, which is a potent inhibitor for proton-pump ATPase, blocked reaction-product formation along the ruffled-border membrane, in mitochondria, and in cytoplasmic vesicles at alkaline pH, but not at neutral pH. It is concluded that the alkaline pH method for Mg+ +-ATPase appears to demonstrate sites of proton-pump ATPase activity.  相似文献   

14.
The specific activity of (Na+ + Mg2+)-dependent ATPase is three times greater in the microsomes of sea-water eels than in freshwater eels; the specific activity is one quarter of that of (Na+ + K+ + Mg2+)-dependent ATPase in both cases.(Na+ + Mg2+)-dependent ATPase is optimally active in a medium containing 8 mM NaCl, 4 mM MgCI2, 4 mM ATP, pH 8.8 and at 30 °C; the enzyme is inhibited by ouabain, by NaCl concentrations > 100 mM and by treatment with urea.It is concluded that the (Na+ + Mg2+)-dependent ATPase activity of gills arises from the presence of a (Na+ + K+ + Mg2+)-dependent ATPase.  相似文献   

15.
Abstract— A comparison was made between K+-Mg2+ activated p-nitrophenyl phosphatase and Na+-K+-Mg2+ activated adenosine triphosphatase with a solubilized enzyme preparation from a membrane fraction of cerebral cortex. The NPPase showed activity even in the absence of phospholipid, whereas the ATPase required the lipid for its activity. More varied types of phospholipids were effective in activating the NPPase than the ATPase, and with each phospholipid the extent and the pattern of the NPPase activation differed from that of the ATPase. By deoxycholate treatment the pH optimum of the NPPase was shifted independently from the pH optimum shift of the ATPase. The specific activity ratio of the NPPase to the ATPase was not constant during purification. These two enzymes were, however, not separable with ammonium sulphate fractionation, and their thermo-lability was identical regardless of the presence of phospholipid. The results suggested two possibilities: (1) the NPPase is a separate enzyme entity from the ATPase; (2) although the NPPase is a part of the ATPase system, the mechanism of action of lipids on the former part differs from that on the rest of the system.  相似文献   

16.
Askerlund P  Evans DE 《Plant physiology》1992,100(4):1670-1681
Purification and functional reconstitution of a calmodulin-stimulated Ca2+-ATPase from cauliflower (Brassica oleracea L.) is described. Activity was purified about 120-fold from a microsomal fraction using calmodulin-affinity chromatography. The purified fraction showed a polypeptide at 115 kD, which formed a phosphorylated intermediate in the presence of Ca2+, together with a few polypeptides with lower molecular masses that were not phosphorylated. The ATPase was reconstituted into liposomes by 3-([cholamidopropyl]-dimethylammonio-)1-propanesulfonate (CHAPS) dialysis. The proteoliposomes showed ATP-dependent Ca2+ uptake and ATPase activity, both of which were stimulated about 4-fold by calmodulin. Specific ATPase activity was about 5 μmol min−1 (mg protein)−1, and the Ca2+/ATP ratio was 0.1 to 0.5 when the ATPase was reconstituted with entrapped oxalate. The purified, reconstituted Ca2+-ATPase was inhibited by vanadate and erythrosin B, but not by cyclopiazonic acid and thapsigargin. Activity was supported by ATP (100%) and GTP (50%) and had a pH optimum of about 7.0. The effect of monovalent and divalent cations (including Ca2+) on activity is described. Assay of membranes purified by two-phase partitioning indicated that approximately 95% of the activity was associated with intracellular membranes, but only about 5% with plasma membranes. Sucrose gradient centrifugation suggests that the endoplasmic reticulum is the major cellular location of calmodulin-stimulated Ca2+-pumping ATPase in Brassica oleracea inflorescences.  相似文献   

17.
Between pH 4–10, basal ATPase activity, measured in the absence of mineral ions, was 10 to 100 times higher in the final cytoplasmic supernatant from potato tuber homogenates than in the membraneous fractions (purified plasmalemma, purified mitochondria and microsomes). The soluble ATPase was slightly inhibited, whereas the membrane-bound ATPases were all stimulated by Mg2+ ions. A further stimulation by Na+ or K+ ions was only observed in purified plasmalemma or mitochondria, at alkaline pH (7.5–9.5). At a fixed (Na++ K+) concentrations (80 mM), this last stimulation was much greater in purified mitochondria (350%) than in plasmalemma (33%); it also increased with (Na++ K+) concentrations up to 200 mM in mitochondria whereas, in plasmalemma, it was roughly constant for monovalent ion concentrations between 20 and 200 mM. General properties of the plasma membrane-bound ATPase have been determined, i.e. substrate specificity, activity variations with quantity of substrate, temperature, pH, etc. Divalent cations stimulated strongly the ATPase in the following order: Mn2+ > Mg2+ > Ca2+. The maximum ATP hydrolysis velocity for that part of ATPase activity which is strictly dependent on Mg2+ ions was 3.85 μmol × mg?1 protein × h?1. This plasma membrane ATPase was not sensitive to ouabaïn or to oligomycin.  相似文献   

18.
R B Koch  D Desaiah 《Life sciences》1975,17(8):1315-1320
The sensitivity of fire ant, Solenopsis richteri (Forel), head homogenate ATPase to its venom and to a cyclohexane extract of whole fire ants were investigated. Na+K+ and oligomycin-sensitive Mg2+ ATPase activities were inhibited by both preparations. Oligomycin-insensitive Mg2+ ATPase activity was inhibited by low concentrations but showed strong stimulation at high concentrations of the venom preparations. Lineweaver-Burk plots of enzyme data in the presence or absence of inhibitor indicated that the inhibitor action was non-competitive with ATP for Na+K+ and oligomycin-sensitive Mg2+ ATPase activities. However, the oligomycin-insensitive Mg2+ ATPase activity showed a mixed type response to the inhibitor. Tests on pure samples of known venom components indicate that they cause the observed effects on the ATPase activities.  相似文献   

19.
ATPase activity was investigated in phloem-containing tissuesof Ricinus communis in relation to its proposed role in phloemloading. Cytochemical staining of cotyledons revealed an ATP-hydrolysingactivity on the plasma membrane of the sieve tube/companioncell complex. Microsomal fractions prepared from cotyledonsand main veins contained a Mg2+-dependent ATPase activity whichshowed low stimulation by KC1 particularly at pH 6.5. The pHoptimum was at pH 8.5 to 90, although the effect of azide indicatedthe presence of mitochondrial ATPase. At pH 6.5, the cited optimumfor plasma membrane ATPase, the activity showed strong inhibitionby PCMBS, vanadate and DCCD. A high pyrophosphatase activitywas observed at pH 8.5. Acidification of the medium by intactcotyledons was increased by fusicoccin and inhibited by PCMBS,NEM and vanadate. Proton pumping by microsomal vesicles as measuredby quinacrine fluorescence was also inhibited by PCMBS, NEMand vanadate. Sucrose uptake by cotyledon discs showed stronginhibition by PCMBS, NEM and CCCP but was little affected byvanadate. Sucrose uptake varied with the developmental stageof the cotyledons and this correlated with microsomal ATPaseactivity measured at pH 6.5, although the precise cellular originof this activity is not certain. The results are discussed inrelation to the role of ATPase activity and proton pumping inphloem loading. Key words: ATPase, phlocm loading, proton pumping, Ricinus communis, sucrose  相似文献   

20.
The properties of membrane-associated ATPase of cucumber (Cucumis sativus cv. Seiriki No. 2) roots cultured in a complete medium (complete enzyme) and in a medium lacking Ca2+ (Ca2+-deficient enzyme) were investigated. The basal activity of membrane-associated ATPase increased during Ca2+ starvation, while Mg2+-activation of the enzyme decreased and even resulted in inhibition by high Mg2+ concentration at the late stage of the Ca2+ starvation. The complete enzyme had low basal activity and showed a Mg2+-activated hyperbolic reaction curve in relation to ATP concentration. Ca2+-deficient enzyme with high basal activity showed a biphasic reaction curve and Mg2+-activation was seen only at high ATP concentrations. Activation of membrane-associated ATPase by various cations was decreased or lost during Ca2+ starvation. The basal ATPase activity of Ca2+-deficient enzyme increased for various substrates including pyrophosphate, p-nitrophenyl phosphate, glucose-6 phosphate, β-glycerophosphate, AMP, ADP and ATP. Mg2+-activation was found only for ADP and ATP in both the complete and Ca2+-deficient enzymes, but the activation for ATP was greatly reduced by Ca2+ starvation. The heat inactivation curves for basal and Mg2+-activated ATPase did not differ much between the complete and Ca2+-deficient enzyme. The delipidation of membrane-associated enzyme by acetone affected the protein content and the basal activity slightly, but inhibited the Mg2+-activated ATPase activity clearly with somewhat different behaviour between the complete and Ca2+-deficient enzyme.  相似文献   

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