Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.6.1.3 (ATPase)
 65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The purified 20,000-dalton fragment of sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase has been shown by us (A.E. Shamoo, T.E. Ryan, P.S. Stewart, D.H. MacLennan, 1976. J. Biol. Chem. 251:4147) to have Ca2+-selective ionophoric activity. The Ca2+-ionophoric fragment has been purified by either SDS-column chromatography or SDS-preparative gel electrophoresis. The Ca2+-ionophoric fragment has been subjected to prolonged dialysis to insure the removal of bound SDS from the fragment. The selectivity sequence of this fragment in black lipid membranes (BLM) formed from either oxidized cholesterol or phosphatidylcholine/cholesterol is the same, PBa greater than PCa greater than PSr greater than PMg greater than PMn. This selectivity sequence is the same as that for the intact (Ca2+ + Mg2+)-ATPase. Treatment of the fragment with cholate to absolutely insure the removal of bound SDS resulted in the fragment having a selectivity sequence as above except that PMn greater than PMg. This and other data indicate that the 20,000-dalton fragment is the site containing the Ca2+-ionophoric activity of the (Ca2+ + Mg2+)-ATPase.
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PMID:Ionophorous properties of the 20,000-dalton fragment of (Ca2+ + Mg2+)-ATPase in phosphatidylcholine: cholesterol membranes. 15 58

A method for primary culture of ovine myometrial cells is described. After dissection, myometrium of ewe uteri was digested in trypsin and collagenase. The cells were preplated for 1 h at 37 degrees C. The non-attached cells were grown in appropriate medium supplemented with 2% fetal calf serum. They had a doubling time of 3 days, reached confluency at 10 days and did not exhibit contact inhibition. Cultures were maintained up to 22 days. Characterization of the cells was achieved by electron microscopy, analysis of myosin in cell extracts and assessment of hormone sensitivity. The cells were found to contain myofilaments, characteristic of smooth muscle. A high content of myosin (6--13%) was demonstrated on SDS-polyacrylamide gel electrophoresis: this was confirmed by ATPase activity assay. Cells responded to estradiol stimulation by increased protein synthesis, and bound [3H]estradiol in a specific and saturable way. These results suggest that myometrial cells grown in primary culture should provide a useful model for studying the hormonal control of contractile protein synthesis.
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PMID:Myometrial cells in primary culture: characterization and hormonal profile. 15 21

A pure, enzymatically active Ca2+-dependent adenosine triphosphatase (Ca2+-ATPase) has been isolated from canine ventricular sarcoplasmic reticulum. In contrast to that derived from skeletal muscle, the Ca2+-ATPase from cardiac sarcoplasmic reticulum was more active when solubilization and subsequent purification took place in the presence of its substrates, Ca2+ and ATP. Cholate- or deoxycholate-solubilized Ca2+-ATPase is recovered following rapid glycerol dilution and centrifugation. The Ca2+-ATPase is stable and possesses hydrolytic capacities up to 4 mumol/mg/min. Sodium dodecyl sulfate-polyacrylamide gels reveal the presence of one protein in the range of 95,000 to 100,000 daltons. This method also yields purified Ca2+-ATPase from fast skeletal muscle of similar activities to those reported by other laboratories.
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PMID:Rapid purification of canine cardiac sarcoplasmic reticulum Ca2+-ATPase. 15 59

1. The subunit compositions of the F1 (oligomycin-insensitive) and F1--F0 (oligomycin-sensitive) mitochondrial ATPase complexes from Saccharomyces cerevisiae have been examined by the highly resolving technique of sodium dodecyl sulphate-polyacrylamide slab gel electrophoresis using a discontinuous buffer system. When isolated in the presence of protease inhibitors, F1 and F1--F0 contained five and twelve bands, respectively; this contrasts with the four- and ten-band patterns seen previously using the less resolving disc gel method. When isolated in the absence of protease inhibitors both F1 and F1--F0 contain spurious polypeptides produced by proteolytic modification. 2. Endogenous protein turnover in S. cerevisiae was impaired in the presence of protease inhibitors. F1--F0 isolated from cells grown in the presence and absence of inhibitors contained an identical polypeptide composition, suggesting that the subunits are not significantly modified by endogenous proteases prior to cell harvesting. 3. Yeast F1--F0 prepared in the presence of protease inhibitors contains a latent, sodium dodecyl sulphate-activated protease contaminant. Sodium dodecyl sulphate-induced proteolysis is largely confined to the 52 000 dalton alpha subunit which degrades into polypeptides of 40 000 and 10 700 daltons. The 40 000 dalton band is apparently equivalent to the polypeptide previously designated subunit 3. 4. Both F1 and F1--F0 were isolated from Torulopsis glabrata, a yeast with considerably shorter mitochondrial DNA than that in S. cerevisiae. F1--F0 catalysed high rates of ATP--32Pi exchange when reconstituted into phospholipid vesicles, thus demonstrating the presence of a complete coupling mechanism. F1--F0 contained approximately twelve subunits and F1 five, like the S. cerevisiae complexes. It therefore appears that the shorter mitochondrial DNA length does not produce a significantly simpler ATPase subunit structure.
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PMID:The yeast mitochondrial ATPase complex. Subunit composition and evidence for a latent protease contaminant. 15 54

The Ca-regulatory system in squid mantle muscle was studied. The findings were as follows. (a) Squid mantle myosin B (squid myosin B) was Ca-sensitive, and its Ca-sensitivity was unaffected by addition of a large amount of rabbit skeletal myosin (skeletal myosin) or rabbit skeletal F-actin (skeletal F-actin). (b) Squid myosin was prepared from the mantle muscle. It showed a heavy chain component and two light chain components in the SDS-gel electrophoretic pattern: the molecular weights of the latter two were 17,000 and 15,000. Actomyosin reconstituted from squid myosin and skeletal (or squid) actin showed Ca-sensitivity in superprecipitation and Mg-ATPase assays. EDTA- treatment had no effect on the Ca-sensitivity of squid myosin. (c) Squid mantle actin (squid actin) was prepared by the method of Spudich and Watt. Hybrid actomyosin reconstituted by using the pure squid actin preparation with skeletal myosin showed no Ca-sensitivity in Mg-ATPase assay, whereas that reconstituted using crude squid actin showed marked Ca-sensitivity. The crude squid actin contained four protein components which were capable of associating with F-actin in 0.1 M KCl, 1 mM MgCl2 and 20 mM Tris-maleate (pH7.5). (d) Native tropomyosin was prepared from squid mantle muscle, and it conferred Ca-sensitivity on skeletal actomyosin as well as on a hybrid actomyosin reconstituted from squid actin and skeletal myosin. (e) Squid native tropomyosin was separated into troponin and tropomyosin fractions by placing it in 0.4 M LiCl at pH 4.7. The troponin fraction was further purified by DEAE-cellulose chromatography. Squid troponin thus obtained was different in mobility from rabbit skeletal or carp dorsal troponin; three bands of squid troponin corresponded to molecular weights of 52,000, 28,000, and 24,000 daltons. It could confer Ca-sensitivity in the presence of tropomyosin on skeletal actomyosin as well as on a hybrid reconstituted from squid actin and skeletal myosin. (f) Squid myosin B, and two hybrid actomyosins were compared as regards Ca and Sr requirements for their Mg-ATPase activities. The myosin-linked regulatory system rather than the thin-filament-linked regulatory system was predominant in squid myosin B. Squid myosin B required higher Ca2+ and Sr2+ concentrations for Mg-ATPase activity; half-maximal activation of Mg-ATPase was obtained at 0.8 micron Ca2+ and 28 micron Sr2+ with skeletal myosin B, and at 2.5 micron Ca2+ and 140 micron Sr2+ with squid myosin B.
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PMID:Two calcium regulation systems in squid (Ommastrephes sloani pacificus) muscle. Preparation of calcium-sensitive myosin and troponin-tropomyosin. 15 2

ATP-induced transport by fractions of frog gastric microsomes prepared either by density gradient centrifugation of by free flow electrophoresis were K+ dependent and hence considered due to a K+-activated ATPase. Significant activity of this enzyme was, however, only found in the anodic peak of the free flow electrophoretic separation, which in addition to separating transporting from non-transporting particles, also separated membranes containing a phosphorylatable peptide (Mr=105 000) region as the major peptide on SDS-polyacrylamide gel electrophoresis from those containing a peptide (Mr=44 000) on SDS-polyacrylamide gel electrophoresis. H+ uptake, measured either by acridine orange or 3,3'-diethyloxadicarbocyanine + tetrachlorosalicylanilide absorbance changes was dependent on K+ intravesicularly. Using 86Rb+, active extrusion of the cation followed ATP addition. SCN-, an inhibitor of acid secretion did not affect the latter, but blocked signals due to H+ uptake, in contrast to mammalian preparations.
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PMID:Transport characteristics of frog gastric membranes. 15 47

A myosin-like protein was extracted and partially purified from a flowering plant, Egeria densa. It had no p-nitrophenyl phosphatase activity, but exhibited EDTA(K+)-ATPase [EC 3.6.1.3] activity at high ionic strength. Its molecular weight as estimated by gel filtration was 4-5 X 10(5). The presence of a heavy chain (MW = about 1.8 X 10(5)) was indicated by SDS-gel electrophoresis. Egeria myosin aggregated in an environment of low ionic strength and formed bipolar filaments. It bound with skeletal muscle F-actin with a periodicity of 40 nm.
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PMID:Identification of myosin in a flowering plant, Egeria densa. 15 12

1. Crayfish (Procambarus clarki) myosin was obtained from abdominal flexor muscle. The Ca2+-ATPase activity of crayfish myosin was much lower than that of rabbit skeletal myosin. However, F-actin-activated Mg2+-ATPase of crayfish and its superprecipitation closely resembled those of rabbit skeletal myosin. This fact suggests that the ability of crayfish myosin to combine with F-actin is essentially the same as that of skeletal myosin, although the chemical structures of both the myosin molecules when involved in their Ca2+-ATPast activity must be different from each other. 2. Crayfish and rabbit skeletal myosins were subjected to SDS-polyacrylamide gel electrophoresis. Crayfish myosin was found to have one heavy chain and two distinct light chain components (CF-gl and CF-g2), which have molecular weights of 18,000 and 16,000, respectively. These light chains correspond in molecular weight to the light chains (SK-g2 and SK-g3) in rabbit skeletal myosin. 3. CF-g1 could be liberated from the crayfish myosin molecule reacting with 5,5'-dithio-bis (2-nitrobenzoic acid), (Nbs2), without recovery of ATPase activity by the addition of DTT. These properties are equivalent to those of SK-g2 in rabbit skeletal myosin, although Nbs2-treated crayfish myosin did not recover its ATPase activity at all.
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PMID:Myosin from abdominal flexor muscle in a crayfish, Procambarus clarki Girard. 15 14

Isolation of a microsomal fraction from human gastric mucosa followed by density gradient centrifugation yielded a vesicular membrane preparation free of mitochondrial markers, containing a K+-activated, ouabain-insensitive ATPase with an activity of 20.7 mumol P1 released/mg protein per h. Sodium dodecyl sulfate gel electrophoresis showed that the human gastric membrane vesicles contained a major polypeptide of 110,000 daltons, which accounted for approximately or equal to 30% of the total protein stained and was phosphorylated by [gamma-32P]ATP and dephosphorylated in the presence of K+. Electron microscopy revealed the presence of vesicles with an average size of 0.13 micrometer in diameter. Addition of 0.65 microM ATP to this vesicular preparation resulted in the uptake of 17 nmol H+/mg protein which was dependent on the presence of K+. The gradient was dissipated by a combination of valinomycin and protonophore after consumption of the ATP. Incubation of fixed human fundic sections or human gastric biopsy with monospecific hog gastric membrane antibody followed by fluorescein-conjugated goat anti-rabbit gamma-globulin, showed fluorescent staining in the middle portion of the gastric glands. These data indicate that human stomach contains a H+ transport ATPase with characteristics similar to those established for lower species.
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PMID:An acid transporting enzyme in human gastric mucosa. 15 36

The latent coupling factor (F1)-ATPase of Micrococcus lysodeikticus has been purified to homogeneity as determined by a number of criteria including, nondenaturing polyacrylamide gel electrophoresis, crossed immunoelectrophoresis and analytical ultracentrifugation. By inclusion of 1 mM phenylmethyl sulfonyl fluoride, a serine protease inhibitor, in the shock-wash step of release of F1 from the membranes, the spontaneous activation of both crude and purified ATPase by endogenous membrane protease(s) can be prevented, thereby yielding a highly latent ATPase preparation. Equilibrium ultracentrifugation of the latent ATPase gave a molecular weight of 400 000. The ATPase contained five different subunits alpha, beta, gamma, delta, and espsilon and their molecular weights determined by SDS-polyacrylamide gel electrophoresis were 60 000, 54 000, 37 000, 27 000 and 9000, respectively. The subunit composition was determined with 14C-labelled, F1-ATPase prepared from cells grown on medium containing [U-14C]-labelled algal protein hydrolysate. Within the limitations of this method the results tentatively suggest a subunit composition of 3 : 3 : 1 : 1 : 3.
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PMID:Purification and properties of the latent F1-APTase of Micrococcus lysodeikticus. 15 61


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