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Query: EC:3.6.3.1 (Mg2+-ATPase)
 1,484 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Various reaction intermediates of sarcoplasmic reticulum Ca2+,Mg2+-ATPase were stabilized and accumulated by modifying a specific SH group or by using nucleotide analogs. Conformational changes of the Ca2+,Mg2+-ATPase during the catalytic cycle were studied in the stabilized intermediates by the use of fluorescent and spin probes, which were introduced at specific SH groups of ATPase, namely one highly reactive but functionally nonessential (SHN) and one essential for the decomposition of the E-P intermediate (SHD) [Kawakita, M., et al. (1980) J. Biochem. 87, 609-617]. The fluorescence intensity of N-(7-dimethylamino-4-methyl-3-coumarinyl)maleimide attached to SHD decreased by 2.5% upon addition of 10 microM AMP-P(NH)P provided that Ca2+ was also present. The AMP-P(NH)P-induced fluorescence change could also be detected by using other fluorescent probes such as N-[p-(2-benzimidazolyl)phenyl]maleimide and N-(1-anilinonaphthyl-4)maleimide. Moreover, labeling at SHN gave similar results. When SHN was labeled with N-[p-(2-benzimidazolyl)phenyl]maleimide, the fluorescence intensity also decreased by 2.5% upon addition of ATP only in the presence of Ca2+, where E-P formation took place. A conformational difference between ECa1-P X ADP and ECa1-P was suggested from saturation transfer ESR measurement of spin-labeled ATPase by using ADP beta S as an ADP analog to cause accumulation of ECa1-P X ADP beta S complex. Possible structural similarities among some of the intermediates are discussed based on these findings.
J Biochem 1983 Sep
PMID:Studies on conformational transitions of Ca2+,Mg2+-adenosine triphosphatase of sarcoplasmic reticulum. II. Conformational characteristics of stabilized reaction intermediates as revealed by fluorescent and paramagnetic probes. 613 71

The effects of tertiary amine local anesthetics (procaine, lidocaine, tetracaine and dibucaine) and chlorpromazine were investigated for three enzyme activities associated with rat brain synaptosomal membranes, i.e., (Na+ + K+)-ATPase (ouabain-sensitive), Mg2+-ATPase (ouabain-insensitive) and acetylcholinesterase. Approximately the same concentrations of each agent gave 50% inhibition of both ATPases, for example 7.9 and 10 mM tetracaine for Mg2+-ATPase and (Na+ + K+)-ATPase, respectively; these concentrations are 10-fold higher than required for inhibition of mitochondrial F1-ATPase. The relative inhibitory potency of the several agents was proportional to their octanol/water partition coefficients. Acetylcholinesterase was inhibited by all agents tested, but the ester anesthetics (procaine and tetracaine) were considerably more potent than the others after correction for partition coefficient differences. For tetracaine, 0.18 mM gave 50% inhibition and showed competitive inhibition on a Lineweaver-Burk plot, but for dibucaine a mixed type of inhibition was observed, and 0.63 mM was required for 50% inhibition. Tetracaine evidently binds at the active site, and dibucaine at the peripheral or modulator site, on this enzyme.
Biochim Biophys Acta 1984 Sep 07
PMID:Inhibition of synaptosomal enzymes by local anesthetics. 614 63

It has been possible to specifically label rabbit skeletal muscle actin at Lys-237 with 2,4-pentanedione, producing an enamine. This reaction can be reversed with hydroxylamine. The modification can be carried out with actin in either the G- or F-forms and does not affect polymerization-depolymerization. The modification does affect, however, the interaction of tropomyosin (Tm) with the modified F-actin. In the absence of Ca2+ and Mg2+ (mu = 0.12), Tm failed to bind to the modified F-actin whereas it did bind to unmodified F-actin (1 Tm:7 actins). Tm binding could be restored under these conditions by the addition of either troponin (Tn), Mg2+, or Mg2+ and Ca2+. Under certain conditions, Tm alone has been shown to inhibit actin-activated heavy meromyosin (HMM)-Mg2+-ATPase. This inhibition did not occur with the modified F-actin even though Tm was bound (approximately 1 Tm:7 actins). Even when Tn was added to this system (in the absence of Ca2+), no inhibition of ATPase could be observed. Thus, this modification appears to prevent F-actin X Tm from assuming the "blocking" inhibitory position (conformation). In addition, Tn appears to enhance the activation of heavy meromyosin-Mg2+-ATPase by the modified F-actin X Tm complex whether Ca2+ is present or not. This state may be analogous to the potentiated state (Murray, J. M., Knox, M. K., Trueblood, C. E., and Weber, A. (1982) Biochemistry 27, 906-915) seen with myosin subfragment 1-saturated actin at low ATP levels. Thus, using modified and unmodified F-actin, it is possible to produce three Tm X actin states: off (F-actin X Tm), on (modified F-actin X Tm), and "potentiated" (modified F-actin X Tm X Tn).
J Biol Chem 1984 Sep 10
PMID:Modification of Lys-237 on actin by 2,4-pentanedione. Alteration of the interaction of actin with tropomyosin. 614 49

The interaction between polymerized tubulin from porcine brain and myosin from rabbit skeletal muscle was examined. The addition of myosin to the solution of tubulin polymerized by taxol resulted in a remarkable increase in turbidity within a few minutes at 37 degrees C, and a dense and stable precipitate was formed. The maximal molar ratio of tubulin bound to myosin was calculated to be about 4, while the value was about 2 when 6S tubulin was used. Both podophyllotoxin and colchicine suppressed the taxol-dependent increase of the binding of tubulin to myosin, but only when they were preincubated with tubulin prior to addition of taxol. 6S tubulin inhibited with actin-activated Mg2+-ATPase activity of myosin, and polymerized tubulin inhibited the Mg-ATPase more than 6S tubulin. Dense precipitates of tubulin and myosin were observed by thin-section electron microscopy. Microtubules were observed to be entangled in myosin filaments and single microtubules were occasionally surrounded by five myosin filaments in a cross section, similar to actin-myosin arrays in muscle. After incubation of tubulin with myosin, taxol was able to induce tubulin polymerization in the same way as it polymerized microtubules in the absence of myosin.
Can J Biochem Cell Biol 1984 Sep
PMID:Effect of taxol on the interaction of tubulin with myosin filaments. 614 2

Solubilization of rat liver lysosome membranes with octyl glucoside or lauryl sarcosinate and analysis of ATPase activities in sections of polyacrylamide gels after electrophoresis revealed one major peak at pH 8 and two peaks at pH 5. The pH 8 ATPase peak was not localized in the same peak with pH 5 ATPase activity, suggesting that these were catalyzed by different proteins. Ca2+- and Mg2+-ATPase activities at pH 8 were present in the same major peak, with Ca2+ activity predominating. The pH 8 Ca2+-ATPase was also not present in the same area of the gels as Ca2+-ADPase.
Biochim Biophys Acta 1984 Sep 27
PMID:Separation of rat liver lysosome membrane adenosine triphosphatase activities by polyacrylamide gel electrophoresis. 620 91

The effects of trypsin digestion and low temperature on Ca2+ binding and on Ca2+ activation of ATP hydrolysis by the high-affinity transport sites of the Ca2+, Mg2+-ATPase of sarcoplasmic reticulum were examined. Sarcoplasmic reticulum vesicles contain 0.7-1.1 high-affinity Ca2+ sites per 10(5) g sarcoplasmic reticulum with K = 3-5 X 10(5) M-1, as well as sites of lower affinity. The first cleavage of the ATPase with trypsin (TD1) has no effect on the binding properties of the high affinity sites. The second tryptic cleavage (TD2) decreases the affinity of the high sites to K = 3 X 10(4) M-1 with conservation of the total number of sites. The purified ATPase contains 1.6-2.0 high affinity Ca2+ sites per 10(5) g protein when measured at 23 degrees C, while at 0-4 degrees C there is approximately equal to 1 high-affinity (K = 5 - 10 X 10(5) M-1) affinity site and approximately equal to 1 intermediate-affinity (K = 3 X 10(4) M-1) site per 10(5) g. Trypsin digestion to the point of TD1 has no effect on either the number or the binding constants of the high-affinity sites. Upon TD2 cleavage, one of the sites is converted to the intermediate-affinity state, while the other remains at high affinity. After TD2 modification of the enzyme both of the sites are in the intermediate affinity state at 4 degrees C. On the basis of the binding data, several models for the roles of the Ca2+ sites in the activation of ATP hydrolysis are derived. The results are summarized by a scheme in which the two high-affinity Ca2+ sites are heterogeneous with respect to sensitivity to temperature and to TD2 modification. The results of this and a previous study [Scott, T. L. and Shamoo, A. E. (1982) J. Membr. Biol. 64, 137-144] indicate that while occupation of either of the two Ca2+ sites can stimulate ATP hydrolysis, the site which is sensitive to TD2 is essential for the coupling of hydrolysis to Ca2+ transport.
Eur J Biochem 1984 Sep 03
PMID:Distinction of the roles of the two high-affinity calcium sites in the functional activities of the Ca2+-ATPase of sarcoplasmic reticulum. 623 83

Two distinctly different ATPases have been reported to be endogenous to the mitotic apparatus: a Mg2+-ATPase resembling axonemal dynein, and a Ca2+-ATPase postulated to be bound in membranes. To examine the nature of the Mg2+-ATPase, we isolated membrane-free mitotic spindles from Stronglylocentrotus droebachiensis embryos by rapidly lysing these in a calcium-chelating, low-ionic-strength buffer (5 mM EGTA, 0.5 mM MgCl2, 10 mM PIPES, pH 6.8) that contained 1% Nonidet P-40. The fibrous isolated mitotic spindles closely resembled spindles in living cells, both in general morphology and in birefringence. In electron micrographs, the spindles were composed primarily of microtubules, free from membranes and highly extracted of intermicrotubular cytoplasmic ground substance. As analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), the pelleted spindles contain 18% tubulin, variable amounts of actin (2-8%), and an unidentified protein of 55 kdaltons in a constant weight ratio to tubulin (1:2.5). The isolated spindles also contained two polypeptides, larger than 300 kdaltons, that comigrated with egg dynein polypeptides, and ATPase activity (0.02 mumol Pi/mg . min) that closely resembled both flagellar and egg dynein. The spindle Mg2+-ATPase showed a ratio of Ca2+-/Mg2+-ATPase = 0.85, had minimal activity in KCl and EDTA, and cleaved GTP at 35% of the rate of ATP. The Mg2+-ATPase was insensitive to ouabain or oligomycin. The spindle Mg2+-ATPase was inhibited by sodium vanadate but, like egg dynein, was less sensitive to vanadate than flagellar dynein. The spindle Mg2+-ATPase does not resemble the mitotic Ca2+-ATPase described by others. We propose that the spindle Mg2+-ATPase is egg dynein. Bound carbohydrate on the two high-molecular-weight polypeptides of both egg dynein and the spindle enzyme suggest that these proteins may normally associate with membranes in the living cell.
J Cell Biol 1980 Sep
PMID:Dynein-like Mg2+-ATPase in mitotic spindles isolated from sea urchin embryos (Strongylocentrotus droebachiensis). 644 5

The defective coupling factor F1 ATPase from a mutant strain (KF11) of Escherichia coli was purified to a practically homogeneous form. The final specific activity of Mg2+-ATPase was 6-9 units/mg protein, which is about 10-15 times lower than that of F1 ATPase from the wild-type strain. The mutant F1 had a ratio of Ca2+-ATPase to Mg2+-ATPase of about 3.5, whereas the wild-type F1 had ratio of about 0.8. The mutant F1 was more unstable than wild-type F1: on storage at -80 degrees C for 2 weeks, about 80% of its activity (dependent on Ca2+ or Mg2+) was lost, whereas none of the activity of the wild-type F1 was lost. The following results indicate that the mutation is in the beta subunit. (i) High Mg2+-ATPase activity (about 20 units/mg protein) was reconstituted when the beta subunit from wild type F1 was added to dissociated mutant F1 and the mixture was dialyzed against buffer containing ATP and Mg2+. (ii) Low ATPase activity having the same ratio of Ca2+-ATPase to Mg2+-ATPase as the mutant F1 was reconstituted when a mixture of the beta subunit from the mutant F1 and the alpha and gamma subunits from wild-type F1 was dialyzed against the same buffer. (iii) Tryptic peptide analysis of the beta subunit of the mutant showed a difference in a single peptide compared with the wild-type strain.
J Biochem 1980 Sep
PMID:Coupling factor F1 ATPase with defective beta subunit from a mutant of Escherichia coli. 644 52

Rabbit liver actin and its associated proteins were prepared and their properties were studied. Liver cells were isolated from excised rabbit liver after perfusion in situ with calcium-free Lock's solution. Dried powder of acetone-treated liver cells was extracted with a buffer previously used to extract actin from skeletal muscle. The liver actin was recovered by adding skeletal myosin to trap actin as actomyosin and the resulting complex was purified by centrifugation. The actin was then dissociated from myosin by adding MgATP and was purified by centrifugation. This fraction showed the characteristic properties of F-actin and was composed of 42K, 53K, and 61K proteins. Further fractionation of these proteins into three components was carried out by centrifugation, DNase-1 affinity chromatography, and preparative gel electrophoresis. The 42K protein proved to be actin since it activated the myosin Mg2+-ATPase activity, interacted with DNase-1, and had a very similar amino acid composition to skeletal muscle actin. In these experiments, binding affinity among these proteins was apparent. Analysis of subcellular fractions combined with the above results indicated that the liver cell 53K and 61K proteins were not soluble fraction components in the cytosol. The physicochemical properties of 53K and 61K proteins were compared with those of gizzard desmin, a typical intermediate filament protein.
J Biochem 1983 Sep
PMID:Actin and actin-associated proteins of rabbit liver cell. 660 66

Intact secretory vesicles isolated from bovine adrenal medulla contain 94 nmol Na+ per mg of protein, and Ca2+ influx into the vesicles is inhibited by increasing concentrations of extravesicular Na+ (but not of K+, Li+ or choline+) or by addition of the Na+ ionophore monensin. Thus Ca2+ influx in determined by the Na+ gradient across the vesicular membrane. Half maximal inhibition of Ca2+ influx occurs with 34 nM Na+ extravesicularly. The fact that Ca2+ can also be released from the vesicles by inversion of the Na+ gradient provides direct evidence that an Na+-Ca2+ exchange may operate. According to an analysis of the inhibition of Ca2+ uptake by Na+ in a Hill plot 2 Na+ would be exchanged for 1 Ca2+. Ca2+ influx into the vesicles increases with temperature (energy of activation; 16 kcal/mol), can be observed already with 10(-7) M free Ca2+ and increases up to 10(-4) M Ca2+. Ca2+ influx is not affected by Mg2+ but Sr2+ is inhibitory. Since the process is only slightly influenced by the pH of the incubation medium and is insensitive to Mg2+-ATP or inhibitors of the proton translocating Mg2+-ATPase the electrochemical proton gradient across the vesicular membrane does not affect directly the Ca2+ influx into the secretory vesicles. Ca2+ uptake is insensitive to ruthenium red and oligomycin.
Biochim Biophys Acta 1982 Sep 24
PMID:Uptake of Ca2+ by isolated secretory vesicles from adrenal medulla. 681 86


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