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 removal of tightly bound nucleotides from mitochondrial F1-ATPase was found to affect the inhibition by ADP and chemical reactivity toward 7-chloro-4-nitro-2,1,3-benzoxadiazole (NBD-C1) and sulfhydryl reagents. Preincubation of nucleotide-depleted F1 with 40 microM ADP in the presence of ethylenediaminetetraacetic acid (EDTA) resulted in a 51% inhibition of the steady-state level of ATPase activity whereas only a 25% inhibition was observed for native F1. Both partially inhibited states of the enzyme could be reversed by the subsequent addition of ATP. Measurement of [14C]ADP binding to nucleotide-depleted F1 in the presence of EDTA reveals three equivalent ADP binding sites with a Kd of 0.45 microM, and a fourth site of lower affinity. The sulfhydryl reagents 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and N-ethylmaleimide (NEM) were found to inhibit the ATPase activity of nucleotide-depleted F1 but not native F1 or nucleotide-depleted F1 in the presence of ADP or ATP. Polyacrylamide gel electrophoresis of nucleotide-depleted F1 labeled with [14C]NEM gave a 2-fold increase in incorporation into the (alpha + beta) subunits and a 7-fold increase in label in the gamma subunit after 90 min compared to when ADP was present during the reaction. ADP binding to the noncatalytic sites enhanced the rate of inhibition of nucleotide-depleted F1 by NBD-C1 about 2-fold while retarding the subsequent intramolecular transfer from an essential phenol group to an amino group about 2.8-fold. The results suggest a conformational change in F1 caused by changes in nucleotide--protein interaction at the noncatalytic sites.
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PMID:Changes in chemical properties of mitochondrial adenosinetriphosphatase upon removal of tightly bound nucleotides. 622 55

Myosin subfragment 1 (S1) heavy chains were prepared from chicken muscle S1 by immunoadsorption in NH4Cl and from rabbit muscle S1 by ion exchange chromatography at 37 degrees C in MgATP. Both heavy chain preparations contained some intact S1, and the ATPase activities of both preparations were less than those of control S1. Recombinations of these heavy chains with alkali light chains prior to removing NH4Cl or MgATP lessened the decreases in ATPase activities. However, once NH4Cl or MgATP was removed, alkali light chain recombination did not result in any increase in ATPase activity. Thus, the lower activities appear to result from the instability of the S1 heavy chain in the absence of alkali light chain. When incubated with a 10-fold molar excess of radiolabeled alkali light chains for 1 h under approximately physiological conditions, almost all of the alkali light chains of S1 but only 8% of those of myosin or myofibrils were exchanged. Aggregation of myosin into filaments accounts for part of this difference in exchangeability. Removal of 30% of the 19,000-Da 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) light chains from myosin increased 3-fold the amount of alkali light chain exchanged. The exchange of the alkali light chains of S1 is inhibited by the presence of DTNB light chains, and cleavage of the DTNB light chains of heavy meromyosin to 17,000-Da fragments increased the rate of alkali light chain exchange. There was no detectable difference in the exchangeability of the two different alkali light chains.
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PMID:Myosin heavy chain-light chain recombinations and interactions between the two classes of light chains. 622 39

In the reaction of sarcoplasmic reticulum membranes with excess 5,5'-dithiobis(2-nitrobenzoate) (DTNB) some new features were observed: The Ca2+-dependent ATPase activities of increasingly modified preparations were considerably enhanced during the initial stage of thiol blockage. A maximum (130-160% of the control activity) was reached when about 1.5-2 mol thiol groups per 10(5) g vesicular protein had reacted, in the absence of ATP and detergent. At higher extents of modification inactivation occurred. Purified ATPase behaved principally similar to native sarcoplasmic vesicles. In the presence of Mg2+ and ATP the activity maximum (up to 180% of control) was broadened and shifted towards a higher degree of thiol blockage. Concomitantly the modification and inactivation rates were considerably reduced. Glycerol (10-30%, v/v) slightly enhanced the ATPase activity maximum and reduced the rate of inactivation essentially only by decreasing the DTNB modification rate. In the presence of sufficient myristoylglycerophosphocholine for solubilization no activation was observed. The steady state level of phosphoprotein from ATP was raised to about 150% of the control level 10 s after addition of DTNB (about 1/2 thiol blocked), followed by a linear decrease with the number of thiols labeled, while the Ca2+-dependent ATPase activity of preparations modified under equivalent conditions (10(-4) M Ca2+ and 2 X 10(-3) M Mg2+ present) showed a broader maximum corresponding to 1.5 thiols blocked.
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PMID:Transient activation of the Ca2+-ATPase from sarcoplasmic reticulum during thiol modification by 5,5'-dithiobis(2-nitrobenzoate). 622 71

Myosins were isolated from the ordinary (white) and dark (red) muscles of yellowtail, Seriola quinqueradiata, and the pH-dependency of ATPase activity, along with some physicochemical properties, was examined. The ordinary muscle myosin contained three kinds of light chain (A1, DTNB and A2 light chains), the molecular weights of which were 28,000, 20,000, and 16,000, respectively. The dark muscle myosin possessed only two kinds of light chain (D1 and D2), the molecular weights of which were 26,000 and 20,000 respectively. These ordinary and dark muscle myosins resemble the fast and slow muscle myosins of the higher vertebrate, respectively, in light chain pattern. The pH optima of the ordinary muscle myosin Ca2+-ATPase activity appeared at 6-6.5 and 9-10, irrespective of whether the enzyme reaction was started by the addition of ATP to the preincubated reaction mixture containing myosin (method I), or vice versa (method II). In the case of the dark muscle myosin, a small peak appeared at around pH 8.5 on the alkaline side when the activity was assayed by method I, whereas a prominent peak appeared at around 9.5 when it was assayed by method II, suggesting instability of this myosin under alkaline conditions. In connection with this, the reaction mixture at pH 9.5 showed a very small and slow increase in turbidity, suggesting a change in the physical state of myosin. The ordinary muscle myosin exhibited approximately three times higher actin-activated Mg2+-ATPase activity than the dark muscle myosin. Superprecipitation activity was also higher in the former than the latter actomyosin. However, both actomyosins showed similar pH-superprecipitation activity profiles.
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PMID:The pH-dependency of myosin ATPases from yellowtail ordinary and dark muscles. 623 Dec 79

Covalent cross-linking reaction between SH1 and SH2 groups in myosin subfragment-1 (S-1) by N,N'-p-phenylenedimaleimide (pPDM) was followed by the degree of inactivation of NH4+-EDTA ATPase activity. The rate of the cross-linking reaction decreased to less than a 20th in the presence of F-actin. The inhibitory effect of F-actin was not observed in the presence of MgATP. Binding of F-actin to S-1 was measured using ultracentrifugation. S-1 whose SH1 and SH2 were covalently cross-linked by pPDM or 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) did not bind F-actin. After the DTNB-cross-linked S-1 is reduced by dithiothreitol, the ability to bind F-actin is recovered. These results suggest that S-1 has a binding site for F-actin in the region between SH1 and SH2. This site appears to determine the high affinity of acto-S-1 complex at the rigor while decreasing the affinity more than 10(2) times in the presence of MgATP.
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PMID:Binding of F-actin to a region between SH1 and SH2 groups of myosin subfragment-1 which may determine the high affinity of acto-subfragment-1 complex at rigor. 623 66

The effect of sulfhydryl reagents on phagocytosis and concomitant enzyme release and on ionophore A 23187 + Ca2+-induced exocytosis in rabbit polymorphonuclear leukocytes (PMN's) was studied. Membrane-penetrating sulfhydryl reagents such as cytochalasin A and N-naphthylmaleimide in micromolar concentrations inhibit both phagocytosis and exocytosis. Poorly penetrating reagents such as p-chloromercuribenzene sulfonate (pCMBS) and 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), inhibit only in high concentrations (pCMBS), or they are ineffective as inhibitors (DTNB). Inhibition by pCMBS is not reversed by glutathione or dithiothreitol; this suggests that some pCMBS probably enters the cell. Specific intracellular sulfhydryl compounds appear to be essential in the cellular apparatus involved in phagocytosis and exocytosis; various possibilities are considered. A concentration of N-naphthylmaleimide which completely inhibits phagocytosis and exocytosis leaves cellular ATPase activity intact.
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PMID:Effects of sulfhydryl reagents on phagocytosis and exocytosis in rabbit polymorphonuclear leukocytes. 624 54

Studies in our laboratory have shown that the anion-sensitive Mg-ATPase is located in mitochondria, but not in the plasma membrane of rabbit gastric mucosa, trout gill, rabbit kidney and rat pancreas; whereas in rabbit erythrocyte membrane, it is part of the Ca-Mg activated ATPase system. These findings appear to rule out a function of the anion-sensitive ATPase in the transport of anions and protons across the plasma membrane in these tissues. On the other hand, the K-activated ATPase in a gradient-purified vesicle fraction of pig gastric mucosa mediates proton uptake in exchange for K+ in the presence of ATP, in agreement with earlier findings of other investigators. The enzyme requires a phospholipid environment for its activity. Studies of arginine modification with butanedione in the presence or absence of ATP and its analogues, and of activating cations indicate that the enzyme contains an essential arginine group involved in ATP binding; and that K+ induces a conformational change, which leads to decreased ATP binding and probably coincides with enzyme dephosphorylation. Similar studies of sulfhydryl modification with DTNB indicate that the enzyme contains an essential sulfhydryl group, which does not appear to be directly involved in ATP binding, but rather that ATP binding may induce a conformational change which makes the sulfhydryl group less accessible.
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PMID:Transport ATPases in anion and proton transport. 624 51

1. (Na+ + K+)-ATPase from rectal glands of Squalus acanthias contains 34 SH groups per mol (Mr 265000). 15 are located on the alpha subunit (Mr 106000) and two on the beta subunit (Mr 40000). The beta subunit also contains one disulphide bridge. 2. The reaction of (Na+ + K+)-ATPase with N-ethylmaleimide shows the existence of at least three classes of SH groups. Class I contains two SH groups on each alpha subunit and one on each beta subunit. Reaction of these groups with N-ethylmaleimide in the presence of 40% glycerol or sucrose does not alter the enzyme activity. Class II contains four SH groups on each alpha subunit, and the reaction of these groups with 0.1 mM N-ethylmaleimide in the presence of 150 mM K+ leads to an enzyme species with about 16% activity. The remaining enzyme activity can be completely abolished by reaction with 5-10 mM N-ethylmaleimide, indicating a third class of SH groups (Class III). This pattern of inactivation is different from that of the kidney enzyme, where only one class of SH groups essential to activity is observed. 3. It is also shown that N-ethylmaleimide and DTNB inactivate by reacting with the same Class II SH groups. 4. Spin-labelling of the (Na+ + K+)-ATPase with a maleimide derivative shows that Class II groups are mostly buried in the membrane, whereas Class I groups are more exposed. It is also shown that spin label bound to the Class I groups can monitor the difference between the Na+- and K+-forms of the enzyme.
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PMID:Sulphydryl groups of (Na+ + K+)-ATPase from rectal glands of Squalus acanthias. Titrations and classification. 628 32

The ATP/ADP exchange is shown to be a partial reaction of the (H+ +K+)-ATPase by the absence of measurable nucleoside diphosphokinase activity and the insensitivity of the reaction to P1, P5-di(adenosine-5') pentaphosphate, a myokinase inhibitor. The exchange demonstrates an absolute requirement for Mg2+ and is optimal at an ADP/ATP ratio of 2. The high ATP concentration (K0.5=116 microM) required for maximal exchange is interpreted as evidence for the involvement of a low affinity form of nucleotide site. The ATP/ADP exchange is regarded as evidence for an ADP-sensitive form of the phosphoenzyme. In native enzyme, pre-steady state kinetics show that the formation of the phosphoenzyme is partially sensitive to ADP while modification of the enzyme by pretreatment with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) in the absence of Mg2+ results in a steady-state phosphoenzyme population, a component of which is ADP sensitive. The ATP/ADP exchange reaction can be either stimulated or inhibited by the presence of K+ as a function of pH and Mg2+.
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PMID:ATP/ADP exchange activity of gastric (H+ +K+)-ATPase. 628 70

The partial purification of (Na+ + K+)-ATPase from pig lens has been achieved by treatment with deoxycholate followed by density gradient centrifugation. The specific activity of the final preparation, ranging from 300 to 500 nmol/h per mg protein, is increased approx. 100-fold compared to the homogenate. A parallel increase in rho-nitrophenylphosphatase activity is also observed. Sodium dodecyl sulfate (SDS) gel electrophoresis reveals six major protein bands, one of which is the 93 kDa alpha subunit of (Na+ + K+)-ATPase which can be phosphorylated by reaction with [gamma-32P]ATP. A second band contains a glycoprotein which displays an apparent molecular weight of 51000 and thus appears to be the beta subunit of the enzyme. The enzyme is sensitive to ouabain with the I50 for (Na+ + K+)-ATPase and rho-nitrophenylphosphatase inhibition being 1.2 and 1.3 microM, respectively. Several agents which inhibit (Na+ + K+)-ATPase from other tissues such as oligomycin, Ca2+, vanadate, N-ethylmaleimide, rho-chloromercuribenzenesulfonic acid (PCMBS) and 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) also inhibit the lens enzyme. Monovalent cations other than K+ are partially effective in activating the (Na+ + K+)-ATPase and rho-nitrophenylphosphatase activities. The K+ congeners were relatively more effective in supporting (Na+ + K+)-ATPase compared to rho-nitrophenylphosphatase activity. Other kinetic properties of the lens enzyme are also comparable to those of the enzyme from other tissues. Utilizing the partially purified membrane bound enzyme, discontinuities in Arrhenius plots of (Na+ + K+)-ATPase activity, rho-nitrophenylphosphatase activity and fluorescence polarization of the fluidity probe, 1,6-diphenyl-1,3,5-hexatriene (DPH), are observed near the physiological temperature of lens. The possible significance of these observations for the mechanism of cataract formation are discussed.
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PMID:Characterization of partially purified (Na+ + K+)-ATPase from porcine lens. 629 83


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