Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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 topographical organization of oligomycin sensitivity conferring protein (OSCP) in the mitochondrial adenosinetriphosphatase (ATPase)-ATP synthase complex has been studied. The accessibility of OSCP to monoclonal antibodies has been qualitatively visualized by using the protein A-gold electron microscopy immunocytochemistry or quantitatively estimated by immunotitration of OSCP in depolymerized or intact membranes. Besides, OSCP cannot be labeled by 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine ([125I]TID) which selectively labels the hydrophobic core of membrane proteins. These observations demonstrate an external location of OSCP on the inner face of the inner mitochondrial membrane. The position of OSCP relative to other peptides of the complex has been analyzed by cross-linking experiments using either zero length N-(ethoxycarbonyl)-2-ethoxydihydroquinoline or 11-A span dimethyl suberimidate cross-linkers in the ATPase-ATP synthase complex. The OSCP cross-linked products were identified either by immunocharacterization with anti-alpha, anti-beta, or anti-OSCP monoclonal antibodies or by their molecular weight. OSCP was cross-linked with either the alpha- or beta-subunits of F1 or to a subunit of Mr 24 000. Other types of cross-linking were obtained by the labeling of OSCP with [cysteamine-35S]-N-succinimidyl 3-[[2-((2-nitro-4-azidophenyl)amino)ethyl]dithio]propionate ([35S]SNAP) and reconstitution of SNAP-OSCP with F1 in urea-treated submitochondrial particles. Under these conditions, OSCP is found to be adjacent to two other peptides of molecular weight close to 30 000. A comparison is made between the topology and the organization of the b-subunit of Escherichia coli and OSCP, suggesting an analogy between OSCP and the hydrophilic part of the b-subunit.
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PMID:Topography of oligomycin sensitivity conferring protein in the mitochondrial adenosinetriphosphatase-ATP synthase. 287 35

The proteolipid subunit of H+-ATPase was labeled by [14C]N,N'-dicyclohexylcarbodiimide in bovine heart mitochondria. The radioactive labeling was followed using various systems of sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). When using discontinuous SDS-PAGE (Laemmli, U.K., 1970, Nature (London) 227, 680-685) a monomeric (Mr 7600 +/- 1500) and a dimeric form (Mr 17,800 +/- 1200) of the proteolipid were detected, while only the monomeric form was found on urea (8 M) containing gels (SDS-PAGE according to Laemmli; or Swank, R. T., and Munkers, K. D., 1971, Anal. Biochem. 39, 462-477). When using SDS-PAGE with Na-Pi buffer (Weber, K., and Osborn, M., 1969, J. Biol. Chem. 244, 4406-4442), only a dimeric form of the proteolipid (Mr 15,000 +/- 1000) was detected. Experimental data indicate that the different patterns of proteolipid separation are related to the presence of the two distinct proteolipid conformations in the SDS solution.
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PMID:Electrophoretic behavior of the H+-ATPase proteolipid from bovine heart mitochondria. 287 22

The membrane sector (F0) of H+-ATPase was prepared by trypsin and urea treatment of F1-F0 and reconstituted with purified F1. The oligomycin sensitivity of the reconstituted F1-F0 complex obtained by treating F1 or F0 with Mg2+ before binding is much higher than that obtained without Mg2+ treatment. The greater change in the intrinsic fluorescence of the reconstituted F1-F0 complex obtained by Mg2+ treatment suggests that conformational changes may occur during the reconstitution. We deduce that Mg2+ binds to membrane lipids, thus decreasing membrane fluidity and changing the physical state of the lipids to provide a suitable microenvironment for conformational changes in F0. The data also suggest that the conformational change in the F0 portion of the F1-F0 complex can be transmitted to the F1 portion, the conformation of which is in turn altered, resulting in the formation of an F1-F0 complex with high oligomycin sensitivity. On the other hand, Mg2+ may act on F1 directly to induce a suitable conformational change which is then transmitted to F0, resulting in the formation of an H+-ATPase with greater sensitivity to oligomycin.
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PMID:On the mechanism of the reconstitution of F1-depleted ATPase complex with purified F1: possible conformational effects. 288 56

The F1 ATPase of Bacillus subtilis BD99 was extracted from everted membrane vesicles by low-ionic-strength treatment and purified by DEAE-cellulose chromatography, hydrophobic interaction chromatography, and anion-exchange high-performance liquid chromatography. The subunit structure of the enzyme was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the absence and presence of urea. In the absence of urea, the alpha and beta subunits comigrated and the ATPase was resolved into four bands. The mobility of the beta subunit, identified by immunoblotting with anti-beta from Escherichia coli F1, was altered dramatically by the presence of urea, causing it to migrate more slowly than the alpha subunit. The catalytic activity of the ATPase was strongly metal dependent; in the absence of effectors, the Ca2+-ATPase activity was 15- to 20-fold higher than the Mg2+ -ATPase activity. On the other hand, sulfite anion, methanol, and optimally, octylglucoside stimulated the Mg2+ -ATPase activity up to twice the level of Ca2+ -ATPase activity (specific activity, about 80 mumol of Pi per min per mg of protein). The F1 ATPase was also isolated from mutants of B. subtilis that had been isolated and characterized in this laboratory by their ability to grow in the presence of protonophores. The specific activities of the ATPase preparations from the mutant and the wild type were very similar for both Mg2+- and Ca2+ -dependent activities. Kinetic parameters (Vmax and Km for Mg-ATP) for octylglucoside-stimulated Mg2+ -ATPase activity were similar in both preparations. Structural analysis by polyacrylamide gel electrophoresis and isoelectric focusing indicated that the five F1 subunits from ATPase preparations from the mutant and wild-type strains had identical apparent molecular weights and that no charge differences were detectable in the alpha and beta subunits in the two preparations. Thus, the increased ATPase activity that had been observed in the uncoupler-resistant mutants is probably not due to a mutation in the F1 moiety of the ATPase complex.
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PMID:Purification and characterization of the F1 ATPase from Bacillus subtilis and its uncoupler-resistant mutant derivatives. 288 51

Liver mitochondria from rats fed ethanol chronically demonstrated a 35% decrease in mitochondrial ATPase activity. Moreover, the ATPase activity was inhibited only 61% by addition of oligomycin. Treatment of mitochondria from ethanol-fed rats with the detergent, Lubrol-WX, caused the release of 36% of the F1 from the resulting inner membrane particles. In comparison, only 5% of the F1 was dissociated when control mitochondria were subjected to the Lubrol treatment. However, when the units of ATPase activity from the supernatant and particles obtained after Lubrol treatment were added together, their sums were equivalent in preparations from control and ethanol-fed animals. Moreover, polyacrylamide gel electrophoresis analyses indicated equal amounts of the alpha + beta subunits of F1 in mitochondria from control and ethanol-fed rats. Reconstitution experiments with urea particles and F1 prepared from both control and ethanol mitochondria revealed a decrease in oligomycin sensitivity which could be attributed to an alteration in the functioning of either the oligomycin sensitivity conferring protein or a membrane sector subunit that interacts with oligomycin. Analysis by reconstitution also demonstrated that there were no ethanol-elicited alterations in the properties of the F1 portion of the ATP synthase complex. These observations indicate that the activity of the ATP synthase complex is altered significantly by ethanol-elicited changes in the functioning of those polypeptides involved in modulating both oligomycin sensitivity and the association of F1 with membrane sector subunits.
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PMID:Ethanol-elicited alterations in the oligomycin sensitivity and structural stability of the mitochondrial F0 . F1 ATPase. 288 57

By incubating soluble F1 of the beef heart mitochondria with radio active 7-chloro-4-nitro-2,1,3-benzoxadiazole (14C)-NBD-C1 in the dark, a labeled F1 with NBD/F1 molar ratio of 1:1 (0.995:1) was obtained, but 98% of the ATPase activity was inhibited. The inactivated ATPase activity of the NBD-labeled F1 may be restored by DTT, suggesting that an essential tyrosine residue of F1 (O-NBD-F1) may be labeled. By exposure of O-NBD-F1 with 0.83 NBD label per F1 molecule and a stable 82.5% inhibition of the ATPase activity even in the presence of DTT. Urea PAGE analysis clearly showed that a 63.4% of the total radio activity was associated with the beta-subunit. In the beta-fraction from N-NBD-F1 only a 19.90-20% of the labeling was detected. It is calculated that F1 of the beef heart mitochondria contains 3 (2.63-2.64) beta-subunits. The results obtained are discussed in connection with the subunit-subunit and site-site interactions and three-site model for F1 catalysis proposal by Boyer and his group.
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PMID:Subunit stoichiometry and interaction of F1-ATPase of beef heart mitochondria. 290 Nov 43

The rate of tryptic digestion of cardiac troponin C (cTNC) has been shown to be dependent on Ca2+ as was noted earlier for skeletal TNC (sTNC). Two representative peptides have been characterized on the basis of amino acid composition and partial amino terminal sequence analysis. Circular dichroism and fluorescence studies monitored their response to the presence of Ca2+. Their ability to form complexes with the ATPase inhibitory subunit of cardiac troponin (cTNI) was determined by urea - polyacrylamide gel electrophoresis and fluorescence experiments. The ability of these peptides to substitute for whole cTNC in restoring the ATPase activity of a partially inhibited synthetic actomyosin system was also explored. The N-terminal peptide 1-88 already contains a large amount of ordered structure, which indicates that the alpha-helices flanking binding site II of cTNC exist independently of Ca2+. Consequently this peptide shows limited increase in structure in the presence of Ca2+. It binds to cTNI independently of the presence of Ca2+ and could substitute for whole cTNC by relaxing the inhibitory effect of cTNI. The C-terminal peptide 103-158 has a low amount of secondary structure in the absence of Ca2+ but this increases dramatically in the presence of this cation. This peptide could only form a stable complex with cTNI in the presence of Ca2+ and was unable to release the inhibitory effect of cTNI.
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PMID:Trypsin digestion of bovine cardiac troponin C in the presence and absence of calcium. 293 34

Troponin was isolated from striated adductor muscles of the "Akazara" scallop (Chlamys nipponensis akazara), and purified in an active form by DEAE-cellulose (Whatman DE52) column chromatography and subsequent gel filtration on Sephacryl S-300. According to sodium dodecyl sulfate-gel electrophoresis and densitometry, Akazara troponin is composed of three components having molecular weights of 52,000, 40,000, and 20,000 in a molar ratio of 1:1:1. The three components were separated from each other by column chromatography in the presence of 6 M urea and 1 mM EDTA on SP-Sephadex C-50 and DEAE-cellulose. The Mr 20,000 component was regarded as troponin C according to the Ca2+-binding properties, which was found to bind 0.7 mol of Ca2+/mol at 0.1 mM Ca2+. The association constant of Ca2+ to troponin C was estimated to be 5 X 10(5) M-1, and was not affected by the addition of 2 mM MgCl2. The Mr 52,000 component appeared to be troponin I, since it inhibited, together with Akazara tropomyosin, both Mg-ATPase and superprecipitation activities of actomyosin reconstituted from rabbit myosin and actin, and the inhibition of the ATPase activity was diminished by the addition of Akazara troponin C. Finally, the Mr 40,000 component appeared to be troponin T, since it co-precipitated with actin-tropomyosin filament and was indispensable with Akazara troponin C and the Mr 52,000 component (troponin I) for conferring the Ca2+ sensitivity to reconstituted actomyosin.
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PMID:Troponin from Akazara scallop striated adductor muscles. 294 90

Ca2+-ATPase molecules were labeled in intact sarcoplasmic reticulum (SR) vesicles, sequentially with a donor fluorophore, fluorescein-5'-isothiocyanate (FITC), and with an acceptor fluorophore, eosin-5'-isothiocyanate (EITC), each at a mole ratio of 0.25-0.5 mol/mol of ATPase. The resonance energy transfer was determined from the effect of acceptor on the intensity and lifetime of donor fluorescence. Due to structural similarities, the two dyes compete for the same site(s) on the Ca2+-ATPase, and under optimal conditions each ATPase molecule is labeled either with donor or acceptor fluorophore, but not with both. There is only slight labeling of phospholipids and other proteins in SR, even at concentrations of FITC or EITC higher than those used in the reported experiments. Efficient energy transfer was observed from the covalently bound FITC to EITC that is assumed to reflect interaction between ATPase molecules. Protein denaturing agents (8 M urea and 4 M guanidine) or nonsolubilizing concentrations of detergents (C12E8 or lysolecithin) abolish the energy transfer. These results are consistent with earlier observations that a large portion of the Ca2+-ATPase is present in oligomeric form in the native membrane. The technique is suitable for kinetic analysis of the effect of various treatments on the monomer-oligomer equilibrium of Ca2+-ATPase. A drawback of the method is that the labeled ATPase, although it retains conformational responses, is enzymatically inactive.
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PMID:Fluorescence energy transfer as an indicator of Ca2+-ATPase interactions in sarcoplasmic reticulum. 295 Sep 38

Molecular composition of Tetrahymena ciliary dynein has been examined by electron microscopy and gel electrophoresis. SDS-urea gel electrophoresis revealed that Tetrahymena 22S dynein contains three (A alpha, A beta, and A gamma) heavy chains whereas 14S dynein contains only one. The molecular masses of 22S and 14S dynein heavy chains were estimated to be approximately 490 and 460 kD, respectively. Electron microscopy of negatively stained specimens showed 22S dynein has three globular heads and thin stalks, whereas 14S dynein consists of a single head. Chymotrypsin digested each of the three 22S dynein heavy chains into large fragments with different time courses. Sucrose density gradient centrifugation separated the digestion products as two peaks. The one with a larger sedimentation coefficient mainly consisted of two-headed particles having binding ability to doublet microtubules, whereas the other with a smaller sedimentation coefficient consisted of only isolated globular particles. Both fractions had ATPase activities. Thus, one active head of 22S dynein can be isolated by chymotrypsin digestion.
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PMID:Chymotryptic digestion of Tetrahymena 22S dynein. I. Decomposition of three-headed 22S dynein to one- and two-headed particles. 295 81


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