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)

Dicyclohexylcarbodiimide (DCCD) specifically inhibits the F1F0-H+-ATP synthase complex of Escherichia coli by covalently modifying a proteolipid subunit that is embedded in the membrane. Multiple copies of the DCCD-reactive protein, also known as subunit c, are found in the F1F0 complex. In order to determine the minimum stoichiometry of reaction, we have treated E. coli membranes with DCCD, at varying concentrations and for varying times, and correlated inhibition of ATPase activity with the degree of modification of subunit c. Subunit c was purified from the membrane, and the degree of modification was determined by two methods. In the "specific radioactivity" method, the moles of [14C]DCCD per total mole of subunit c was calculated from the radioactivity incorporated per mg of protein, and conversion of mg of protein to mol of protein based upon amino acid analysis. In the "high performance liquid chromatography (HPLC) peak area" method, the DCCD-modified subunit c was separated from unmodified subunit c on an anion exchange AX300 HPLC column, and the areas of the peaks from the chromatogram quantitated. The shape of the modification versus inhibition curve indicated that modification of a single subunit c per F0 was sufficient to abolish ATPase activity. The titration data were fit by nonlinear regression analysis to a single hit mathematical model, A = Un(1 - r) + r, where A is the relative activity, U is the ratio of unmodified/total subunit c, n is the number of subunit c per F0, and r is a residual fraction of ATPase activity that was resistant to inhibition by DCCD. The two methods gave values for n equal to 10 by the specific radioactivity method and 14 by the HPLC peak area method, and values for r of 0.28 and 0.30, respectively. Most of the r value was accounted for by the observed dissociation of 15-20% of the F1-ATPase from the membrane under ATPase assay conditions. When the minimal, experimentally justified value of r = 0.15 was used in the equation above, the calculated values of n were reduced to 8 and 11, respectively. The value of n determined here, with a probable range of uncertainty of 8-14, is consistent with, and provides an independent type of experimental support for, the suggested stoichiometry of 10 +/- 1 subunit c per F1F0, which was determined by a more precise radiolabeling method (Foster, D. L., and Fillingame, R. H. (1982) J. Biol. Chem. 257, 2009-2015).
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PMID:H+-ATPase activity of Escherichia coli F1F0 is blocked after reaction of dicyclohexylcarbodiimide with a single proteolipid (subunit c) of the F0 complex. 252 56

1. HMM and S-1 both bind one mol of calcium per mole of head, and a half of the calcium binding was diminished upon magnesium addition (10 mM) at the low affinity site. 2. The Mg-ATPase activity of HMM (without actin) was fully activated by the binding of one mol of calcium bound per mol of HMM. 3. The calcium binding profile to S-1 is the same as that to HMM, however, the Mg-ATPase activity of S-1 is independent of calcium binding. It is suggested that there are two kinds of myosin head (or S-1) in molluscan myosin, functionally different in calcium binding properties.
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PMID:Calcium binding and calcium-sensitivity of heavy meromyosin and subfragment-1 from squid (Todarodes pacificus) mantle and scallop (Patinopecten yessoensis) adductor muscles. 252 74

The reactive thiol of the myosin head, SH-1, can be selectively labelled in glycerinated rabbit muscle fibres. This residue has been used as an attachment site for either fluorescent or spectroscopic probes which report on head movements and orientations in various functional states of muscle. We have specifically modified SH-1 in vitro, using purified rabbit myosin and conditions similar to those employed in the labelling of muscle fibres (low ionic strength [40 mM NaCl] at 4 degrees C), with stoichiometric amounts of either [14C]-iodoacetamide, 5-(2[iodoacetyl)amino)ethyl) aminonaphthalene-1- sulphonic acid (IAEDANS), or 4-(2-iodoacetamido-2,2,6,6-tetramethyl piperidinooxyl (IASL). The specificity of modification was determined by measuring the well-defined alterations in the high salt ATPase activities of myosin and by localizing both IAAm and IAEDANS to the 20-kDa C-terminal subfragment 1 (S1) which contains SH-1. The low ionic strength actin-activated Mg2+-ATPase of SH-1-modified rabbit myosin was measured in the presence of the thin filament regulatory, complex, troponin-tropomyosin. A significant increase in this activity in the absence of calcium, concomitant with a decrease in activity in the presence of calcium, was observed as the extent of SH-1 modification was incrementally increased from zero to one mole of label bound per mole of SH-1. The elevated myosin Mg2+-ATPase, which results from SH-1 modification, does not account for the increased actin-activated Mg2+-ATPase in resting conditions (i.e. in the absence of calcium).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:SH-1 modification of rabbit myosin interferes with calcium regulation. 252 9

The characteristics and specificity of inactivation of the chloroplast F1-ATPase (CF1) with 7-chloro-4-nitrobenzofurazan (Nbf-Cl) have been investigated. Inactivation of the octylglucoside-dependent Mg2+-ATPase activity of latent CF1 by Nbf-Cl can be correlated with the formation of about 1.2 mol of Nbf-O-Tyr per mole of enzyme. Following inactivation of CF1 with [14C]Nbf-Cl, polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed that the majority of the radioactive reagent incorporated is present in the beta subunit. Treatment of the enzyme with [14C]Nbf-Cl following dithiothreitol heat activation, led to similar labeling of the beta subunit and substantial incorporation of 14C into the gamma subunit. On complete inactivation, about 4 mol of Nbf-S-Cys is formed per mole of dithiothreitol-heat-activated CF1. Incorporation of 14C into the gamma subunit is prevented by prior treatment of the latent CF1 or of the dithiothreitol-heat-activated CF1 with iodoacetamide. Following incubation of the dithiothreitol-heat-activated CF1 with iodoacetamide, complete inactivation of the octylglucoside-dependent Mg2+-ATPase activity by Nbf-Cl can be correlated with the formation of about 1.2 mol of Nbf-O-Tyr per mole of enzyme. After stabilization of the [14C]Nbf-O-Tyr derivative by treatment with sodium dithionite, a labeled peptide was purified. Automatic Edman degradation of this peptide revealed the sequence V-X-V-P-A-D-(D). The majority of the radioactivity was cleaved in the second cycle, the position occupied in CF1 by Tyr-beta-328, which is homologous to Tyr-beta-311, the residue reactive with Nbf-Cl in the beef heart mitochondrial F1-ATPase. When CF1, modified at Tyr-beta-328 with Nbf-Cl, is incubated at pH 9.0, the Nbf-O-Tyr adduct is hydrolyzed, leading to concomitant recovery of the ATPase activity. In double labeling experiments, two-dimensional isoelectric focusing in the presence of urea followed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicates that 2-azido-ADP, covalently bound at the tight ADP binding site, and the tyrosine modified by [14C]Nbf-Cl are located in different beta subunits.
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PMID:Selectivity of modification when latent and activated forms of the chloroplast F1-ATPase are inactivated by 7-chloro-4-nitrobenzofurazan. 252 17

Adenosinetriphosphopyridoxal (AP3PL) specifically modifies Lys684 of Ca2(+)-ATPase of sarcoplasmic reticulum (SR-ATPase) in the presence of Ca2+, leading to its inactivation (Yamamoto, H. et al. (1988) J. Biochem. 103, 452-457). We have now investigated the effects of AP3PL on SR-ATPase in the absence of Ca2+. Similarly to its action in the presence of Ca2+, AP3PL inhibited the Ca2(+)-transporting activity in a dose-dependent manner in the absence of Ca2+ as well. ATP and ADP protected SR-ATPase against inactivation by this reagent. One mole of AP3PL was bound per mol of SR-ATPase with concomitant loss of the Ca2(+)-transporting activity. Binding of AP3PL to SR-ATPase was prevented by ATP. AP3PL-labeled SR membranes were digested with thermolysin and labeled thermolytic peptides were purified through C18 reversed-phase HPLC. Two major AP3PL-labeled peptides were obtained in approximately 1:1 ratio; one was an octapeptide corresponding to 679-ValGluProSerHisLys*SerLys-686, and the other, a nonapeptide corresponding to 487-PheSerArgAspSerLys*ArgMetSer-495 (Lys* indicates a labeled Lys residue) of SR-ATPase. Lys684 in the former turned out to be the same as the highly specific target of AP3PL in the presence of Ca2+ which was identified previously. The target site specificity of AP3PL thus changed significantly but not entirely on binding of Ca2+ to SR-ATPase. This indicates that the spatial arrangement around the gamma-phosphoryl group of the bound ATP is affected by Ca2+ ions bound at the transport site. It is also likely that Lys492 and Lys684 are situated close together in the ATP binding site of SR-ATPase.
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PMID:Ca2(+)-dependent conformational change of the ATP-binding site of Ca2(+)-transporting ATPase of sarcoplasmic reticulum as revealed by an alteration of the target-site specificity of adenosine triphosphopyridoxal. 253 25

The thermotropic behavior of both sulfatide (3-sulfogalactosylceramide) and galactosylceramide in dielaidoylphosphatidylcholine (DEPC) liposomes was studied, using steady-state fluorescence polarization of parinaric acid isomers. The glycosphingolipid (GSL) concentration of the liposomes was varied from 0 to 100%, and phase diagrams were constructed. The data indicate that sulfatide and DEPC are immiscible in the gel phase at sulfatide mole ratios of less than 0.30. The temperature of onset of the gel-liquid-crystalline phase transition is higher in K+ -containing buffer than in osmotically equal Na+ -containing buffer. Similar measurements, using galactosylceramide, a neutral GSL, indicated that this lipid and DEPC are immiscible in the gel phase at galactosylceramide mole ratios of less than 0.40. In contrast to the results obtained with sulfatide, onset temperatures are identical in Na+- or K+-containing buffers. The phase properties of sulfatide/DEPC mixtures are shown to depend on the cation only when the sulfatides contain hydroxy fatty acids. Our observations indicate that physiologically relevant concentrations of monovalent cations affect motion and distribution of sulfatide in biological membranes and further implicate this GSL as an important determinant of function of the Na+,K+-ATPase. A preliminary report of these data [Rintoul, D.A., Welti, R., & Song, W. (1988) Biophys. J. 53, 126a].
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PMID:Thermotropic behavior of mixtures of glycosphingolipids and phosphatidylcholine: effect of monovalent cations on sulfatide and galactosylceramide. 253 58

Two strains of Bacteroides intermedius BH20/30 and BH18/23, have been grown in anaerobic continuous culture under various conditions for periods up to 54 days. Strain BH20/30 grew over a relatively wide pH range from 5-8 with a maximum at pH 7.0 at a dilution rate (D) of 0.1 h-1 with a glucose limitation, while strain BH18/23 had an optimum between 5.8 and 7.3 and would not grow above and below this range. The maximum growth rate (mu max) for the latter strain was shown to be 0.23 h-1, or a doubling time of 3.0 at the upper limit of pH 7.3. The yield values (Y glucose) for strain BH18/23 reached 187-177 g cells (dry weight) per mole of glucose in the optimum pH range (6.0-7.0) and amino acid analysis of the spent medium indicated that these high values were the results of the combined use of glucose and amino acids; the cultures also exhibited proteolytic activity. The major acid end-products in the same pH range were formate and succinate with lesser concentrations of acetate, isovalerate and fumarate; small amounts of lactate appeared as the cells were stressed at pH values above 7.5 when the culture was 'washing out' of the chemostat. Glucose metabolism appeared to function through the glycolytic pathway in B. intermedius BH18/23 since the glycolytic inhibitors, sodium fluoride and sodium iodoacetate, completely inhibited glucose utilization as did the proton ionophore, gramicidin, and the ATPase inhibitor, N,N1-dicyclohexylcarbodiimide (DCCD). Inhibition by these latter compounds indicated that the saccharolytic Bacteroides utilize proton gradients generated by proton-extruding ATPase (H+/ATPase) to conserve energy.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Growth and metabolic properties of Bacteroides intermedius in anaerobic continuous culture. 276 20

The effect of ethylene glycol on the contractile properties of skeletal muscles was studied using glycerinated rabbit psoas muscle fibers. Measurements were made at an ionic strength of 0.2 M, pH 7.0, and at 10 degrees C. Ethylene glycol reversibly reduced isometric tension, active stiffness, the tension-to-stiffness ratio, and the shortening velocity at zero load (Vo) in a dose-dependent fashion. Ethylene glycol also reduced the Ca sensitivity for contraction. The extent of the reduction in Vo by ethylene glycol was much larger than that in the actomyosin ATPase activity reported by Travers and Hillaire (Eur. J. Biochem. 98, 293-299 [1979]). Although ethylene glycol reduced tension and Vo, the MgATP concentration dependence of these two quantities was almost unaffected. These results suggest that in the presence of ethylene glycol, force produced by crossbridges in the principal force-producing state is reduced and/or the relative population of the attached crossbridges in the low-force state increases. The results also suggest that the reduction in Vo by ethylene glycol is caused not only by a reduction in the actomyosin ATPase activity but also by a reduction in the shortening distance per mole of ATP split.
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PMID:Modification of the contractile properties of rabbit skeletal muscle by ethylene glycol. 276 8

Vanadate has been suggested as an intracellular regulator of (Na+ + K+)-ATPase. To test this hypothesis we examined the stimulatory and inhibitory effects of vanadate on 86--Rb efflux and influx (measurements of the activity of the Na-pump) in rat ileum under conditions of normal, reduced and increased (Na+ + K+)-ATPase activity. The half maximal stimulation of the Rb efflux and the half maximal inhibition of the Rb influx were not different in the three conditions tested. This suggests that vanadate does not have a regulatory effect on the activity of the Na-K-transport enzyme. The vanadate effect seem rather, to be nonspecific in terms of being unrelated, on a mole per mole basis, to the activity of the (Na+ + K+)-ATPase enzyme.
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PMID:The nonspecific nature of the vanadate inhibition of rat ileal (NA,K)-ATPase. 284 70

Lucifer yellow (4-amino-N-[3-(vinylsulfonyl)phenyl]naphthalimide-3,6-disulf onate), a fluorescent probe that can react covalently with sulfhydryl or amino groups, has been used to modify chloroplast coupling factor 1 (CF1). Conditions are described under which Lucifer yellow selectively labels the alpha subunit of CF1 to the extent of about 1 mol of probe per mole of CF1. An especially reactive amino group is apparently labeled, and modification has little effect on the ATPase activity of the enzyme. Lucifer yellow is a useful probe for fluorescence energy transfer measurements. The distances between this probe and fluorescent and absorbing molecules attached to seven specific sites on the beta, gamma, and epsilon subunits were determined. These distances converge to a single location. In addition to providing further information about the structure of CF1, these results suggest that the alpha subunits of CF1 are not structurally equivalent.
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PMID:Selective modification of an alpha subunit of chloroplast coupling factor 1. 285 86


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