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)

Lead (Pb) inhibited K(+)-stimulated para-nitrophenyl phosphatase (K(+)-PNPPase) of rat brain P2 fraction in a concentration-dependent manner with IC50 3.5 microM. Altered pH versus activity demonstrated comparable inhibitions by Pb in buffered acidic, neutral and alkaline pH ranges. Inhibition of enzyme activity was higher at lower temperatures (17-27 degrees C) compared to 37 degrees C. Preincubation of enzyme with sulfhydryl (-SH) agents such as cysteine (Cyst) and dithiothreitol (DTT) but not glutathione (GSH) protected against Pb-inhibition. Uncompetitive type of inhibition with respect to the activation of K+ was indicated by a decrease in Vmax from 16.2 to 8.37 mumoles of para-nitrophenol (PNP)/mg protein/hr and Km from 18.99 to 12.39 mM. Kinetic studies on substrate (p-nitrophenyl phosphate) activation in the presence of Pb (3.5 microM) indicated a significant decrease in Vmax from 8.94 to 4.69 mumoles of PNP/mg protein/hr with no change in Km. Cyst (3 microM) and DTT (10 microM) reversed the Pb-inhibited Vmax from 4.69 to 8.38 and 7.24 mumoles of PNP/mg protein/hr respectively. These results suggest that the critical conformational property of K(+)-PNPPase is sensitive to Pb. The data also indicates that the Pb inhibits Na(+)-K+ ATPase system by interacting with dephosphorylation of the enzyme-phosphoryl complex, while Cyst and DTT protected against Pb-inhibition.
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PMID:Effects of lead on K(+)-para-nitrophenyl phosphatase activity and protection by thiol reagents. 216 61

Mercuric chloride (HgCl2), a neurotoxic compound, inhibited the adenosine triphosphatase (ATPase) system in a concentration-dependent manner. Hydrolysis of ATP was linear with time with or without HgCl2 in the reaction mixtures. Higher inhibition of (Na(+)-K+)ATPase activity by HgCl2 was observed in alkaline (8.0 to 9.0) pH and at lower temperatures (17 to 32 degrees). Activation energy values were increased slightly in the presence of HgCl2. Activation of (Na(+)-K+)ATPase by ATP in the presence of HgCl2 showed a decrease in Vmax from 15.29 to 5.0 mumol of inorganic phosphate (Pi)/mg protein/hr with no change in Km. Similarly, activation of K(+)-stimulated p-nitrophenyl phosphatase (K(+)-PNPPase) in the presence of HgCl2 showed a decrease in Vmax from 3.26 to 1.35 mumols of p-nitrophenol (PNP)/mg protein/hr with no change in Km. K(+)-activation kinetic studies indicated that HgCl2 decreased Vmax from 14.01 to 4.30 mumols Pi/mg protein/hr in the case of (Na(+)-K+)ATPase and from 3.45 to 2.40 mumols PNP/mg protein/hr in the case of K(+)-PNPPase with no changes in Km. Na(+)-activation of (Na(+)-K+)ATPase in the presence of HgCl2 showed a decrease in Vmax from 11.06 to 3.23 mumols Pi/mg protein/hr and an increase in Km from 1.06 to 2.08 mM. Preincubation of microsomes with sulfhydryl (SH) agents dithiothreitol, cysteine and glutathione protected HgCl2-inhibition of (Na(+)-K+)ATPase. The data suggest that HgCl2 inhibited (Na(+)-K+)ATPase by interfering with the dephosphorylation of the enzyme-phosphoryl complex.
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PMID:Effect of mercuric chloride on the kinetics of cationic and substrate activation of the rat brain microsomal ATPase system. 216 72

We have recently reported the primary structures of the three unique peptide inhibitors (SPAI-1, -2, and -3) against Na+, K(+)-ATPase which contained four disulfide bridges in common (Biochem. Biophys. Res. Commun. 164, 496 (1989)). The disulfide connectivities of SPAI were determined by the combination of amino acid analyses with the direct application to a gas-phase sequencer of its proteolytic fragments. The disulfide bond was identified by detection of phenylthiohydantoin derivatives of cystine and its decomposed product dehydroalanine. The four cysteine pairs were disclosed to be Cys20 to Cys49, Cys27 to Cys53, Cys36 to Cys48, and Cys42 to Cys57, all linked by disulfide bridge formation. The allocation pattern of these disulfide bonds was the same as that recently reported for human mucous proteinase inhibitor (EMBO J. 7, 345 (1988], though SPAI showed no proteinase inhibitory activity at all.
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PMID:Four disulfide bonds' allocation of Na+, K(+)-ATPase inhibitor (SPAI). 217 23

In the midgut tissue of the silkworm, Bombyx mori, alkaline phosphatase isozymes, membrane-bound (m-ALP) and soluble (s-ALP) forms are controlled by non-allelic genes on the same chromosome. We purified and characterized both ALPs to elucidate their possible functions and to compare with mammalian ALPs. Both forms were found to be similar Mr = 68,000 in gel permeation chromatography and as a single subunit as a monomer in SDS-polyacrylamide gel electrophoresis with Mr = 58,000 for m-ALP and Mr = 61,000 for s-ALP. The pH optima of ALPs were 10.9 (m-ALP) and 9.8 (s-ALP), and the former was extremely stable even in pH 10-12 which accords with the physiological milieu in Bombyx midgut lumen. Both ALPs had similar substrate specificities. L-cysteine inhibited strongly both ALPs, but inhibitory effects of L-phenylalanine, L-homoarginine, and L-leucine were undetectable for s-ALP and very weak for m-ALP. The antibody raised against purified m-ALP recognized m-ALP but not purified s-ALP and vice versa. Rocket-immunoassay showed that m-ALP was distributed in similar levels along the length of midgut except for the most anterior portion. Seventy percent of s-ALP activity existed in the last one-third of midgut. Immunohistochemical study revealed that the m-ALP was localized at the brush border of columnar cells in the middle and posterior midgut epithelia. In contrast, the s-ALP was localized at the apical surface of goblet cells through the length of midgut. We detected ATPase activity in the purified s-ALP preparation; Mg2+ was essential for the ATPase activity and the activity also increased with KHCO3 but not with KCl. The solubilization test of m- ALP with various agents was attempted and the relationship between m-ALP and the digestive fluid-ALP was discussed.
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PMID:Genetically defined membrane-bound and soluble alkaline phosphatases of the silkworm: their discrete localization and properties. 239 71

The domain structure of rho protein, a transcription termination factor of Escherichia coli, was analyzed by oligonucleotide site-directed mutagenesis and chemical modification methods. The single cysteine at position 202, previously thought to be essential for rho function, was changed to serine or to glycine with no detectable effects on the protein's hexameric structure, RNA-binding ability, or ATPase, helicase, and transcription termination activities. A 151-residue amino-terminal fragment (N1), generated by hydroxylamine cleavage, and its complementary carboxyl-terminal fragment of 268 amino acids (N2) were extracted from NaDod-SO4/polyacrylamide gels and renatured. The N1 fragment binds poly(C) and mRNA corresponding to the rho-dependent terminator sequence trp t', but not RNA unrecognized by rho; hence, this small renaturable domain retains not only the binding ability but also the specificity of the native protein. Uncleaved rho renatures to regain its RNA-dependent ATPase activity, but neither N1 nor N2 exhibits any detectable ATP hydrolysis. Similarly, the two fragments, isolated separately but renatured together, are unable to hydrolyze ATP. Sequence homology to the alpha subunit of the E. coli F1 membrane ATPase, and to consensus elements of other nucleotide-binding proteins, strongly suggests a structural domain for ATP binding that begins after amino acid 164. The implications of discrete domains for RNA and nucleotide binding are discussed in the context of requirements for specific interactions between RNA-binding and ATP-hydrolysis sites during transcription termination.
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PMID:Structure of rho factor: an RNA-binding domain and a separate region with strong similarity to proven ATP-binding domains. 245 28

The (Ca2+ + Mg2+)-ATPase (ATP phosphohydrolase (Ca2+-transporting), EC 3.6.1.38) protein of rabbit skeletal sarcoplasmic reticulum (SR) rapidly incorporated 2 mol of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) per 10(5) g of protein with little change in the Ca2+-dependent ATPase activity. When 2 additional mol of the reagent were bound the Ca2+-ATPase, activity was inhibited. The same pattern was found for modified intact SR and the Ca2+ uptake ability was inhibited. MgATP, CaATP and MgADP protected the Ca2+-ATPase activity concurrent with a decrease of about 1 mol of the NBD group per 10(5) g protein, but the Ca2+ uptake ability was not protected. Calcium alone had no effect on the modification. The modified ATPase protein or SR formed non-serial oligomers or aggregates, but the ATPase protein remained the predominant species present. In the presence of MgATP, oligomer formation was reduced partially but the major changes in the Ca2+-ATPase activity were due to the modification of the ATPase monomer. Thiolysis of the NBD-ATPase protein with dithiothreitol did not restore the Ca2+-ATPase activity, although more than 1 mol of the NBD group was removed from cysteine residues. Cysteine residues were modified in the NBD-ATPase protein or SR when the enzyme activity was inhibited. Trypsin digestion of NBD-SR or its ATPase protein released the A, B, A1, and A2 fragments. The A fragment and its subfragment A2 contained most of the label. Substrate MgATP protection studies showed that the A1 and A2 fragments were involved in maintaining the Ca2+-ATPase activity. Reagent-induced conformational changes of these fragments rather than direct active site group labeling accounted for the loss of ATPase activity.
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PMID:Modification of the (Ca2+ + Mg2+)-ATPase protein of sarcoplasmic reticulum with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole. 252 98

Previous studies from this laboratory (Brooker, R. J., and Slayman, C. W. (1983) J. Biol. Chem. 258, 222-226; Davenport, J. W., and Slayman, C. W. (1988) J. Biol. Chem. 263, 16007-16013) have used the sulfhydryl reagent N-ethylmaleimide (NEM) to define two sites on the Neurospora plasma membrane H+-ATPase: a "fast" site which reacts in several minutes with no loss of enzymatic activity and a "slow" site which reacts in tens of minutes to produce complete inactivation of the enzyme. The slow site is protected when MgATP or MgADP is bound to the catalytic site of the ATPase. The present study demonstrates that the fluorescent reagent 5-[2-iodoacetamido)ethyl)-1-aminonaphthalenesulfonic acid (IAEDANS) can be used to label five of the eight cysteine residues of the Neurospora ATPase (Cys376, Cys409, Cys472, Cys532, Cys545). Tryptic peptides bearing those residues have been purified by high performance liquid chromatography and located within the known primary structure of the ATPase by amino acid analysis and/or sequencing. By pretreating the enzyme with NEM in the presence or absence of MgADP before incubation with IAEDANS, it has been possible to identify the fast NEM site as Cys545 and the slow MgADP-protectable NEM site as Cys532. Both residues lie within the central hydrophilic domain of the protein, close to a highly conserved stretch of amino acids that may be involved in nucleotide binding. However, all five IAEDANS-reactive cysteines can be nearly completely modified by the less bulky sulfhydryl reagent methyl methanethiosulfonate with less than 20% inhibition of enzyme activity; thus, none of the five cysteines can be considered to play a direct role in the reaction cycle of the ATPase.
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PMID:Cysteine 532 and cysteine 545 are the N-ethylmaleimide-reactive residues of the Neurospora plasma membrane H+-ATPase. 252 86

The Neurospora crassa plasma membrane H+-ATPase is rapidly inactivated in the presence of diethyl pyrocarbonate (DEP). The reaction is pseudo-first-order showing time- and concentration-dependent inactivation with a second-order rate constant of 385-420 M-1.min-1 at pH 6.9 and 25 degrees C. The difference spectrum of the native and modified enzyme has a maximum near 240 nm, characteristic of N-carbethoxyhistidine. No change in the absorbance of the inhibited ATPase at 278 nm or in the number of modifiable sulfhydryl groups is observed, indicating that the inhibition is not due to tyrosine or cysteine modification, and the inhibition is irreversible, ruling out serine residues. Furthermore, pretreatment of the ATPase with pyridoxal phosphate/NaBH4 under the conditions of the DEP treatment does not inhibit the ATPase and does not alter the DEP inhibition kinetics, indicating that the inactivation by DEP is not due to amino group modification. The pH dependence of the inactivation reaction indicates that the essential residue has a pKa near 7.5, and the activity lost as a result of H+-ATPase modification by DEP is partially recovered after hydroxylamine treatment at 4 degrees C. Taken together, these results strongly indicate that the inactivation of the H+-ATPase by DEP involves histidine modification. Analyses of the inhibition kinetics and the stoichiometry of modification indicate that among eight histidines modified per enzyme molecule, only one is essential for H+-ATPase activity. Finally, ADP protects against inactivation by DEP, indicating that the essential residue modified may be located at or near the nucleotide binding site.
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PMID:Evidence for an essential histidine residue in the Neurospora crassa plasma membrane H+-ATPase. 252 92

ATPase activity of elementary bodies (EBs) of Chlamydia trachomatis was investigated by using high-resolution 31P nuclear magnetic resonance spectroscopy. ATPase activity was detected in EBs of C. trachomatis serovars A, B, and L2 after treatment with the reducing agents 2-mercaptoethanol and glutathione. ATPase activity was oligomycin sensitive and magnesium ion dependent. EBs heated at 60 degrees C for 10 min or pretreated with Triton X-100 before exposure to 2-mercaptoethanol did not exhibit ATPase activity. Monoclonal antibody to the major outer membrane protein abrogated ATPase activity of EBs, whereas monoclonal antibody to chlamydial lipopolysaccharide only marginally reduced the level of ATPase activity. These findings suggest that EBs possess intrinsic ATPase activity and that cysteine-rich outer membrane proteins of EBs are important in the regulation of ATPase activity. The major outer membrane protein may be the major route through which ATP accesses ATPase.
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PMID:High-resolution 31P nuclear magnetic resonance study of Chlamydia trachomatis: induction of ATPase activity in elementary bodies. 253 Jan 75

5-Iodoacetamidofluorescein (5-IAF) labels the catalytic (alpha) subunit of dog kidney Na,K-ATPase without inhibiting enzymatic activity and is thus a useful fluorescent reporter of enzyme conformation under conditions of enzyme turnover. In this study conditions for labeling a unique sulfhydryl group are described, and this residue is identified in the cDNA-derived sequence. Reaction with iodoacetate (IAA) prior to fluorescent labeling lowers the stoichiometry of 5-IAF incorporation from 2.1 to 1.2 mol/mol alpha beta protomer, and increases the conformationally dependent fluorescence changes by 40-50%, consistent with the elimination of nonspecific labeling. IAA/IAF-enzyme has the same catalytic activity as the IAF-enzyme. In contrast, treatment with iodoacetamide prior to labeling with 5-IAF abolishes all fluorescence responses, although activity is retained. IAA/IAF-enzyme was digested by extensive trypsinolysis, and the fluorescent peptides released from the membrane were purified by high performance liquid chromatography and sequenced. Several fluorescent peptides were found, containing all or part of the sequence Cys-Ile-Glu-Leu-Cys-Cys-Gly-Ser-Val-Lys, corresponding to residues 452-461 in the sheep alpha subunit. The major site of modification is the second of the vicinal cysteine residues, Cys-457. Phenylarsine oxide, a reagent specific for vicinal sulfhydryl groups, prevents fluorophore incorporation, thereby confirming the identification of the IAF site from the sequence data.
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PMID:Identification of the 5-iodoacetamidofluorescein reporter site on the Na,K-ATPase. 253 22


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