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)

Schisanhenol (SAL) had been shown to have potent antioxidant activities. The protective effects of SAL against oxygen radical induced mitochondrial injuries of rat heart and liver were investigated in the present study. The ferrous-cysteine induced mitochondrial lipid peroxidation was significantly inhibited by SAL, while the loss of ATPase activity induced by the lipid peroxidation was prevented. The rigidification of mitochondrial membrane, as well as swelling, lysis and disintegration of the mitochondria induced by ferrous-cysteine were all significantly inhibited by SAL. These results further confirm that SAL possesses antioxidant activity.
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PMID:Protective effect of schisanhenol against oxygen radical induced mitochondrial toxicity on rat heart and liver. 158 68

The topography of rapid equilibrium complexes formed between G-actin and myosin subfragment-1, which are the first kinetic intermediates in the polymerization process into F-acto-S1 filaments, has been probed by chemical cross-linking. In the absence of ATP, cross-linking of G-actin-S1 complexes by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) yielded a major 165-170-kDa and a fainter 200-205-kDa doublet polypeptide. The actin:S1 molar ratio was 1 in the EDC-cross-linked complexes, using either double labeling techniques or the method combining EDC + N-hydroxysuccinimide. Chemical cleavages of the covalently cross-linked complexes by formic acid and N-hydroxylamine (Sutoh, K. (1983) Biochemistry 22, 1579-1585) showed that in the main cross-linked 165-kDa polypeptide, the 1-12 acidic N-terminal region of actin was covalently linked to the lysine-rich region connecting the central 50-kDa domain to the C-terminal 20-kDa domain of S1, as in F-acto-S1 complexes. G-actin, but not F-actin, was covalently cross-linked to S1 by N,N'-paraphenylenedimaleimide (p-PDM). A major 195-kDa and a minor 165-kDa polypeptide were obtained, could be separated from actin and S1 by DEAE-cellulose chromatography, and did not exhibit actin-activated Mg-ATPase activity. Both EDC-cross-linked and p-PDM-cross-linked complexes between G-actin and S1 could be incorporated into F-acto-S1 decorated filaments. The C-terminal cysteine 374 of actin is involved in the p-PDM cross-linked 195-kDa complex. Accordingly, a covalent photocross-linked 200-kDa conjugate was formed between S1 heavy chain and benzophenone-G-actin, obtained by covalent modification of Cys374 by benzophenonemaleimide (Tao, T., Lamkin, M., and Scheiner, C. J. (1985) Arch. Biochem. Biophys. 240, 627-634). These results demonstrate that (i) G-actin-S1 and F-actin-S1 complexes display a large similarity in the EDC-cross-linked electrostatic close contacts and (ii) a change in the environment of Cys374 is linked to the polymerization into F-actin-S1 decorated filaments.
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PMID:Interaction between G-actin and myosin subfragment-1 probed by covalent cross-linking. 162 3

The effects of cysteine conjugates of styrene, e.g. S-1/2-(phenyl-hydroxyethyl) cysteine (PEC) and its N-acetyl derivative (NAPEC) on the transport of p-amino-hippurate (PAH) ion in plasma membranes were studied in vitro using isolated rat renal brush-border membrane (BBM) and basolateral membrane (BLM) vesicles. The uptake of PAH was significantly inhibited by both PEC and NAPEC in both the membrane vesicles, as verified by decrease of the membrane/medium concentration ratio of PAH as the concentration of either PEC or NAPEC in the medium increased. These results show that both PEC and NAPEC are capable of interfering with the accumulation of PAH (a model organic anion for renal tubular transport system) by both energy-independent and energy-dependent carrier-mediated transport processes. The inhibition of PAH uptake in BBM vesicles due to 10 mM PEC or NAPEC was found to be nearly competitive, almost similar to probenecid, whereas in BLM vesicles such inhibition was found to be partially noncompetitive, as verified by the double reciprocal plots. Both PEC and NAPEC showed dose-dependent inhibition of the specific activity of the marker enzyme in each membrane, e.g. gamma-glutamyl transferase in BBM and Na(+)-K(+)-ATPase in BLM vesicles. However, no such inhibition was noticed with probenecid. The in vitro pretreatment with probenecid prevented the inhibition of gamma-glutamyl transferase activity in BBM due to PEC or NAPEC, but such was not the case for the Na(+)-K(+)-ATPase activity in BLM. In conclusion, the data suggest that the transport of cysteine or N-acetylcysteine conjugates of styrene by renal proximal tubular cells across both the membrane vesicles accompanied by the inhibition of the membrane-specific enzymes may lead to cellular dysfunction and consequently to the initial development of their nephrotoxicity.
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PMID:Effects of cysteine derivatives of styrene on the transport of p-aminohippurate ion in renal plasma membrane vesicles. 165 14

N-(1-Pyrene)maleimide is a hydrophobic, sulfhydryl-directed, chemical modification probe which, at a low concentration, inhibits the capacity of lamb kidney sodium- and potassium-activated adenosine triphosphatase [Na,K)-ATPase; EC 3.6.1.3) to bind ouabain. This inhibition is partially blocked by preincubation of the enzyme with ouabagenin, an aglycone derivative which can be used as a reversible protecting ligand for the ouabain binding site. The kinetics of inhibition are not first order, suggesting that there may be more than one site of labeling which is responsible for the inhibition of ouabain binding. Although earlier work (Kirley, T. L., Lane, L. K., and Wallick, E. T. (1986) J. Biol. Chem. 261, 4525-4528) indicates that the inhibition is accompanied by a loss in the number of binding sites rather than a decrease in affinity of the sites for the ligand, other data (Scheiner-Bobis, G., Zimmerman, M., Kirch, V., and Schoner, W. (1987) Eur. J. Biochem. 165, 653-656) indicates that there is no cysteine residue located extracellularly in the ouabain binding site. By sequence analysis of alpha subunit peptides labeled by N-(1-pyrene)maleimide in the absence but not in the presence of protecting ligand, it is demonstrated in this work that there are two major sites of labeling protected by the binding of ouabagenin, Cys-367 and Cys-656. Both of these sites are located in the large cytoplasmic domain of the alpha subunit, one close to the phosphorylation site (Asp-369), and the other implicated in the binding of ATP (Cys-656). Therefore, it appears from this data that the inhibition of ouabain binding by N-(1-pyrene)maleimide is not due to modification of a site in the binding pocket for cardiac glycosides, but rather to an allosteric effect, since cardiac glycoside binding is known to be dependent on the phosphorylation state of the enzyme. The dependence of inhibition on the presence of sodium, potassium, and ATP also is consistent with this interpretation. The work reported here thus explains the apparent paradox posed by the earlier data.
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PMID:Identification of cysteine residues in lamb kidney (Na,K)-ATPase essential for ouabain binding. 165 8

Lead (Pb) inhibited the activities of Na(+)-K+ ATPase (IC50 = 2.0 x 10(-6) M), K(+)-Para-Nitrophenyl phosphatase (PNPPase) (IC50 = 3.5 x 10(-6) M) and [3H]-ouabain binding (IC50 = 4.0 x 10(-5) M) in rat brain P2 fraction. A variable temperature or pH significantly elevated the inhibition of Na(+)-K+ ATPase by Pb in buffered acidic, neutral and alkaline pH ranges. Noncompetitive inhibition with respect to activation of Na(+)-K+ ATPase by ATP was indicated by a variation in Vmax values with no significant changes in Km values at any temperature studied. In the presence of Pb, for Na(+)-K+ ATPase at pH 6.5 and 8.5, Vmax was decreased with an increase in Km values suggesting a mixed type of inhibition. Sulfhydryl agents such as dithiothreitol (DTT) and cysteine (Cyst), but not glutathione (GSH) offered varied levels of protection against Pb-inhibition of Na(+)-K+ ATPase at pH 7.5 and 8.5. The present data suggest that inhibition of Na(+)-K+ ATPase by Pb is both temperature and pH-dependent. These results also indicate that Pb inhibited Na(+)-K+ ATPase by interfering with phosphorylation of enzyme molecule and dephosphorylation of the enzyme-phosphoryl complex and exerted an effect similar to that of SH-blocking agents.
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PMID:Effects of lead on pH and temperature-dependent substrate-activation kinetics of ATPase system and its protection by thiol compounds in rat brain. 166 9

It has been reported recently that the isolated and renatured 23-kDa N-terminal fragment of rabbit skeletal muscle myosin binds tightly to F-actin in an ATP-dependent manner [Muhlrad, A. (1989) Biochemistry 28, 4002-4010]. The binding to actin is of electrostatic nature and may involve a positively charged cluster of residues on the 23-kDa fragment stretching from Arg-143 to Arg-147. An octapeptide containing this positive cluster was synthesized and coupled to BSA through a cysteine residue added to the N-terminus of the peptide. Polyclonal antibody was raised against the BSA-coupled peptide in rabbits which recognized the N-terminal 23-kDa fragment of rabbit skeletal myosin subfragment 1, and a peptide comprised of residues 122-204 of the 23K fragment in Western blots. The purified antibody [IgG and F(ab)] inhibited the actin-activated ATPase activity of S1 without affecting its Mg2(+)- and K+(EDTA)-modulated ATPase activity. Both IgG and F(ab) decreased the binding of S1 to F-actin in a sedimentation assay, and actin inhibited the binding of both IgG and F(ab) to S1 in a competitive binding assay. The cysteine thiol of the synthetic octapeptide was labeled by the fluorescent thiol reagent monobromobimane, and the labeled peptide was found to bind to actin in a sedimentation assay. The results support the possibility that the positively charged Arg-143 to Arg-147 stretch of residues on the 23-kDa fragment participates in actin binding of myosin and may represent an essential constituent of the actin-S1 interface.
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PMID:Antibody directed against the 142-148 sequence of the myosin heavy chain interferes with myosin-actin interaction. 170 18

Selenite (Se) cataract in rabbit lenses was investigated in vitro to define target sites of Se that might be involved in calcium elevation and lens opacification. Experiments in which the anterior or the posterior surface of the lens was exposed to Se showed that anterior exposure led to ionic imbalances and opacification in the whole lens. Posterior exposure to Se (1 mM, 2 hr) had no effect. Se treatment (0.1 mM) of epithelial homogenates led to a 56% loss of thiol (SH) groups, and treatment of lenses cultured in Se led to a 22% loss. Experiments to assess the effects of Se on SH groups of Ca-ATPase showed that the transport enzyme was not affected by the poison. To determine whether this negative finding was due to the lack of accessibility of Se for SH sites in an ordered membrane, Ca-ATPase was also assayed in homogenate preparations treated with Se; still no inhibition of Ca-ATPase activity was observed. Therefore, an alternative explanation of calcium elevation was explored. The passive movement of labeled chloride (36Cl) was found to be twice as fast in Se-treated lenses as it was in control lenses. Measurement of the lens voltage indicated an 18-mV depolarization in Se-treated lenses, suggesting that Se increased membrane permeability. All cataractogenic changes that occurred after Se treatment were irreversible-despite intervention with external application of reduced glutathione or cysteine. This finding suggests that irreversible loss of SH groups in lens membranes is important in maintaining ion homeostasis.
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PMID:Effect of selenite on epithelium of cultured rabbit lens. 182 4

The change in reaction energetics of the bicarbonate-dependent ATPase reaction of Escherichia coli carbamoyl phosphate synthetase has been investigated for two site-directed mutations of the essential cysteine in the small subunit. Cysteine 269 has been proposed to facilitate the hydrolysis of glutamine by the formation of a glutamyl-thioester intermediate. The two mutant enzymes, C269S and C269G, along with the isolated large subunit, exhibit a 2-2.6-fold increase in the bicarbonate-dependent ATPase reaction relative to that observed for the wild type enzyme. In the presence of glutamine the overall enhancement is 3.7 and 9.0 for the C269G and C269S mutant enzymes, respectively. Carboxyphosphate is an intermediate in the bicarbonate-dependent ATPase reaction. The cause of the rate enhancements was investigated by measuring the positional isotope exchange rate in [gamma-18O4] ATP relative to the net rate of ATP hydrolysis. This ratio (Vex/Vchem) is a measure of the partitioning of the enzyme-carboxyphosphate-ADP complex. The partitioning ratio for the mutants is identical within experimental error to that observed for the wild type enzyme. This observation is consistent with the conclusion that the ground state for the enzyme-carboxyphosphate-ADP complex in the mutants is destabilized relative to the same complex in the wild type enzyme. If the increase in the absolute rate of ATP hydrolysis was due to a stabilization of the transition state for carboxyphosphate hydrolysis then the positional isotope exchange rate relative to the chemical hydrolysis rate would have been expected to decrease in the mutants.
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PMID:Alterations in the energetics of the carbamoyl phosphate synthetase reaction by site-directed modification of the essential sulfhydryl group. 182 18

Effects of reactive oxygen intermediates generated by hypoxanthine plus xanthine oxidase on the Ca(2+)-adenosinetriphosphatase (ATPase) of sarcoplasmic reticulum from bovine aortic smooth muscle were studied. Exogenous hypoxanthine (0.1-100 microM) plus xanthine oxidase (10 mU/ml) produced an hypoxanthine concentration-dependent inhibition of the Ca(2+)-ATPase. The inhibition could be completely blocked by superoxide dismutase (100 U/ml) but not by either mannitol (20 mM) or deferoxamine (100 microM). Direct addition of hydrogen peroxide in the micromolar range did not cause significant inhibition. These results suggest that superoxide is the primary damaging species. Cysteine blocked this inhibition, suggesting possible involvement of sulfhydryl groups in the inhibition mechanism. Additionally, 1.16 +/- 0.17 mU/g wet wt of xanthine oxidase activity was detected in the postnuclear supernatant of bovine aortic smooth muscle, suggesting the existence of a possible intracellular source of superoxide. This value was calculated to be approximately 5 mU/ml by using a usual value of vascular smooth muscle cellular volume. Thus the level of endogenous xanthine oxidase in vascular smooth muscle is comparable with the level of exogenous xanthine oxidase used in the present study. These findings suggest a potential role of xanthine oxidase-generated superoxide in oxidative damage to vascular smooth muscle during a number of pathophysiological conditions.
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PMID:Inhibition of Ca(2+)-ATPase of vascular smooth muscle sarcoplasmic reticulum by reactive oxygen intermediates. 183 1

Treatment of a solution of actin and smooth muscle caldesmon with 5,5'-dithiobis(2-nitrobenzoic acid) results in the formation of a disulfide cross-link between the C-terminal penultimate residue Cys-374 of actin and Cys-580 in caldesmon's C-terminal actin-binding region. Therefore, these 2 residues are close in the actin-caldesmon complex. Since myosin also binds to actin in the vicinity of Cys-374 and since caldesmon inhibits actomyosin ATPase activity by the reduction of myosin binding to actin, then the inhibition might be by caldesmon sterically hindering or blocking myosin's interaction with actin. [Ca2+]Calmodulin, which reverses the inhibition of the ATPase activity, decreases the yield of the cross-linked species, suggesting a weakening of the caldesmon-actin interaction in the cross-linked region. It is possible to maximally cross-link one caldesmon molecule/every three actin monomers, in the absence or presence of tropomyosin, clearly ruling out an elongated, end-to-end alignment of caldesmon on the actin filament in vitro, and raising the possibility that the N-terminal part of caldesmon projects out from the filament. Reaction of 5,5'-dithiobis(2-nitrobenzoic acid)-modified actin with caldesmon leads to the same disulfide cross-linked product between actin and caldesmon Cys-580, enabling the specific labeling of the other caldesmon cysteine, residue 153, in the N-terminal part of caldesmon with a spectroscopic probe.
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PMID:Disulfide cross-linking of caldesmon to actin. 183 43


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