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Query: UMLS:C0027960 (mole)
 21,279 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In order to improve our understanding of membrane protein solubilization by sodium dodecylsulphate, sarcoplasmic reticulum vesicles have been treated with this surfactant at different detergent: protein mole ratios. Effects on Ca2(+)-ATPase activity, membrane protein solubilization, and protein conformation have been independently monitored, and correlations among the various parameters have been observed. The thermal denaturation of sarcoplasmic reticulum proteins in the presence of sodium dodecylsulphate has also been characterized spectroscopically.
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PMID:Solubilization of sarcoplasmic reticulum membranes by sodium dodecylsulphate. A Fourier-transform infrared spectroscopic study. 214 30

ATP-binding sites in the unphosphorylated Ca2(+)-ATPase of sarcoplasmic reticulum vesicles were titrated with 2',3'-O-(2,4,6-trinitrocyclohexadienylidene)-[3H]AMP (TNP-AMP) or -[3H]ATP (TNP-ATP) in the absence of Ca2+ at pH 7.0 and 0 degrees C by using a centrifugation procedure. In some measurements, the bound TNP-nucleotides were chased with ATP. The data were analyzed by best-fit computer programs as well as by Scatchard plots. The results showed the existence of 1 mol of TNP-AMP binding sites with high affinity (Kd = 7.62 nM) per mole of phosphorylatable sites. The affinity of these sites for ATP (Kd = 10.1 microM) agreed with that of catalytic sites for ATP in the absence of Ca2+. The results further showed the existence of 2 mol of TNP-ATP binding sites with uniform affinity (Kd = 156 nM) per mole of phosphorylatable sites. Half of the bound TNP-ATP was fully chased by low concentrations of ATP. The affinity of this class of the sites for ATP (Kd = 8.9 microM) again agreed with that of catalytic sites for ATP. The other half of the bound TNP-ATP was fully chased only by much higher concentrations of ATP. Thus, the affinity of this class of the sites for ATP (Kd = 791 microM) was much lower than that of catalytic sites for ATP. Similar measurements were performed with sarcoplasmic reticulum vesicles pretreated by N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine. Although the affinities for TNP-ATP and for ATP were appreciably altered by this pretreatment, the results were essentially the same as those obtained with native vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Existence of a low-affinity ATP-binding site in the unphosphorylated Ca2(+)-ATPase of sarcoplasmic reticulum vesicles: evidence from binding of 2',3'-O-(2,4,6-trinitrocyclohexadienylidene)-[3H]AMP and -[3H]ATP. 214 72

Previous studies of titratable (Na+ + K+)-ATPase sulfhydryl groups have indicated the presence of one disulfide bond per mole of holoenzyme. This single disulfide cross-link was assigned to the beta subunit on the basis of the difference between the number of titrated "free" sulfhydryl groups and the total number of titrated sulfhydryl groups for each subunit [Esmann, M. (1982) Biochim. Biophys. Acta 688, 251; Kawamura, M., & Nagano, K. (1984) Biochim. Biophys. Acta 694, 27]. In the present study, beta-subunit tryptic peptides containing disulfide cross-links were identified and purified by HPLC. Two new peptides were generated from each disulfide-bonded peptide by reduction with dithiothreitol, and the amino acid compositions of these reduced peptides were determined. The data demonstrate that there are three disulfide bonds in the native beta subunit: 125Cys-148Cys, 158Cys-174Cys, and 212Cys-275Cys. The number of disulfide bonds in the beta subunit was also estimated by titration of sulfhydryl groups with [14C]iodoacetamide. Six sulfhydryl groups were identified: two sulfhydryl groups were titrated without prior reduction, and four were identified only after reduction of the protein with dithiothreitol. These data, suggesting that the beta subunit contains two disulfide bonds, are inconsistent with the peptide isolation experiments, which directly identified three disulfide bonds in the beta subunit. This inconsistency was resolved by demonstrating that approximately 20% of each disulfide bond in the beta subunit was reduced prior to the start of the experiment, resulting in an underestimation of the number of disulfide-bonded sulfhydryl groups in the beta subunit from the titration experiments.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Beta subunit of (Na+ + K+)-ATPase contains three disulfide bonds. 215 40

The binding and conformational properties of the divalent cation site required for H+,K(+)-ATPase catalysis have been explored by using Ca2+ as a substitute for Mg2+. 45Ca2+ binding was measured with either a filtration assay or by passage over Dowex cation exchange columns on ice. In the absence of ATP, Ca2+ was bound in a saturating fashion with a stoichiometry of 0.9 mol of Ca2+ per active site and an apparent Kd for free Ca2+ of 332 +/- 39 microM. At ATP concentrations sufficient for maximal phosphorylation (10 microM), 1.2 mol of Ca2+ was bound per active site with an apparent Kd for free Ca2+ of 110 +/- 22 microM. At ATP concentrations greater than or equal to 100 microM, 2.2 mol of Ca2+ were bound per active site, suggesting that an additional mole of Ca2+ bound in association with low affinity nucleotide binding. At concentrations sufficient for maximal phosphorylation by ATP (less than or equal to 10 microM), APD, ADP + Pi, beta,gamma-methylene-ATP, CTP, and GTP were unable to substitute for ATP. Active site ligands such as acetyl phosphate, phosphate, and p-nitrophenyl phosphate were also ineffective at increasing the Ca2+ affinity. However, vanadate, a transition state analog of the phosphoenzyme, gave a binding capacity of 1.0 mol/active site and the apparent Kd for free Ca2+ was less than or equal to 18 microM. Mg2+ displaced bound Ca2+ in the absence and presence of ATP but Ca2+ was bound about 10-20 times more tightly than Mg2+. The free Mg2+ affinity, like Ca2+, increased in the presence of ATP. Monovalent cations had no effect on Ca2+ binding in the absence of ATP but dit reduce Ca2+ binding in the presence of ATP (K+ = Rb+ = NH4 + greater than Na+ greater than Li+ greater than Cs+ greater than TMA+, where TMA is tetramethylammonium chloride) by reducing phosphorylation. These results indicate that the Ca2+ and Mg2+ bound more tightly to the phosphoenzyme conformation. Eosin fluorescence changes showed that both Ca2+ and Mg2+ stabilized E1 conformations (i.e. cytosolic conformations of the monovalent cation site(s)) (Ca.E1 and Mg.E1). Addition of the substrate acetyl phosphate to either Ca.E1 or Mg.E1 produced identical eosin fluorescence showing that Ca2+ and Mg2+ gave similar E2 (extracytosolic) conformations at the eosin (nucleotide) site. In the presence of acetyl phosphate and K+, the conformations with Ca2+ or Mg2+ were also similar. Comparison of the kinetics of the phosphoenzyme and Ca2+ binding showed that Ca2+ bound prior to phosphorylation and dissociated after dephosphorylation.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Calcium binding to the H+,K(+)-ATPase. Evidence for a divalent cation site that is occupied during the catalytic cycle. 216 18

1. In order to determine the stoichiometry of K+ uptake and ATP consumption by Na+, K(+)-ATPase in the isolated, perfused mandibular gland of the rat, oxygen consumption and net K+ uptake from the vascular side were measured during the recovery period following K+ depletion by stimulation with acetylcholine in combination with ouabain. 2. Acetylcholine (10(-6) M) induced fluid secretion and an initial, transient release of K+ from the gland. Addition of ouabain suppressed salivary fluid secretion and caused an additional, dose-dependent, transient release of K+. 3. With acetylcholine (10(-6) M), the oxygen consumption of the gland increased to 62.4 +/- 4.2 microliters/(g min) from a resting value of 12.9 +/- 1.2 microliters/(g min). The increased oxygen consumption was suppressed by ouabain, in a dose-dependent fashion. 4. Withdrawal of acetylcholine and ouabain induced a net uptake of K+ and, simultaneously, an increase in oxygen consumption. The cumulative K+ uptake and the increment of oxygen consumption during the recovery period were dependent on the concentration of used ouabain. 5. The rates of K+ uptake and ATP hydrolysis were compared during recovery, assuming that six moles of ATP were hydrolysed for each mole of oxygen consumed. The data obtained during the later phase of the recovery lay on a single straight line with a slope of 1.8 for each of the various concentrations of ouabain, suggesting that the relationship between K+ uptake and energy consumption was linear. 6. Assuming the K+:ATP stoichiometry of the Na+, K(+)-ATPase to be 2:1, K+ would appear to be transported with ca 90% efficiency by Na+, K(+)-ATPase in the rat mandibular gland. Using 1.8-2.0 as a K+/ATP stoichiometry, the rate of primary active K+ uptake and the corresponding passive K+ efflux during secretion were estimated to be 20-22 mumol/(g min) from the oxygen consumption and the net K+ flux. 7. The passive K+ efflux was estimated, from the initial rate of K+ release caused by addition of 10(-3) M-ouabain, to be 30 mumol/g min). The discrepancy between the estimated active K+ uptake and passive K+ release (8-10 mumol/(g min] could be attributed to a secondary active K+ uptake process such as Na(+)-K(+)-2Cl-symport.
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PMID:Oxygen consumption for K+ uptake during post-stimulatory activation of Na+, K(+)-ATPase in perfused rat mandibular gland. 217 14

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


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