Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.6.1.3 (ATPase)
 65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The fluorogenic sulfhydryl probe 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM) (1-50 nM) is used to characterize the functional role and location of highly reactive thiol groups on the ryanodine-sensitive Ca2+ release channel complex [i.e., ryanodine receptors (RyRs)] of skeletal and cardiac junctional sarcoplasmic reticulum (SR). The kinetics of forming fluorescent CPM adducts with junctional but not longitudinal SR membrane proteins (0.02-1 pmol of CPM/microgram of SR protein) are found to be markedly dependent on the presence of physiological and pharmacological modulators of the RyR Ca2+ channel. RyR agonists, micromolar Ca2+, and nanomolar ryanodine promote a slow SR thiol-CPM reaction, with an apparent rate constant k of 0.0021 +/- 0.0002 sec-1, and > 89% of the fluorescence is associated with the 110-kDa Ca2+ pump, which constitutes 68% of the protein in the SR preparations. However, in the presence of Ca2+ channel antagonists (millimolar Mg2+, millimolar Ca2+, or micromolar ryanodine), CPM rapidly forms adducts with a single class of highly reactive (hyperreactive) SR thiols (k = 0.025 +/- 0.002 sec-1). Nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis of CPM-labeled SR protein and Western blot analyses with antiryanodine or antitriadin antibodies reveal that the hyperreactive thiols labeled by CPM under conditions favoring channel closure are localized principally to the RyR protomer and triadin, which constitute < 6% of the protein in the SR preparation. Immunoprecipitation experiments with antiryanodine and antitriadin monoclonal antibodies confirm the location of CPM-labeled thiol groups on RyR and triadin, respectively. The results indicate that the RyR and triadin contain a small number of highly reactive cysteine residues that selectively conjugate with CPM only when channel closure is favored. It is shown that either 1) the redox state (sulfhydryl/disulfide status) or 2) the accessibility of the hyperreactive thiols on the RyR and triadin is determined by the conformational state of the channel. Covalent modification of hyperreactive thiols with nanomolar CPM inhibits both Ca(2+)-induced Ca2+ release and the gating activity of single channels reconstituted in bilayers, revealing the essential functional importance of hyperreactive thiols on channel-associated proteins. 1,4-Naphthoquinone (0.4-40 pmol/micrograms of protein) selectively oxidizes hyperreactive thiols on RyR and triadin and releases Ca2+ from SR vesicles, without inhibiting Ca(2+)-ATPase activity. The results provide direct evidence of the existence and functional role of hyperreactive cysteine residues on the RyR and triadin in regulating the gating of ryanodine-sensitive intracellular Ca2+ channels and strongly suggest that these important Ca2+ regulatory channels may be an important target for oxidative cell damage mediated by quinones.
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PMID:Direct evidence for the existence and functional role of hyperreactive sulfhydryls on the ryanodine receptor-triadin complex selectively labeled by the coumarin maleimide 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin. 811 70

Na,K-ATPase in right-side-out oriented vesicles was stabilized in different conformations, and the location of intramembrane Cys residues of the alpha-subunit was assessed with membrane-permeable and membrane-impermeable Cys-directed reagents. In the presence of Mg2+ and Pi, Cys964 was the most accessible for both membrane-impermeable 4-acetamido-4'-maleimidylstilbene-2, 2'disulfonic acid (or stilbene disulfonate maleimide, SDSM) and membrane-permeable 7-diethylamino-3-(4'-maleimidyl)-4-methylcoumarin (CPM). In the presence of K+, Cys964 was modified only by hydrophobic CPM, indicating that the environment around Cys964 was different in these two conformations. Cys964 seems to mark the extracellular border of transmembrane segment M9. Cys911 in transmembrane segment M8 showed similar behavior; however, it was not so readily modified. Complete modification of Cys964 and Cys911 causes only partial (about 50%) inactivation of both ATPase activity and Rb+ (or K+) occlusion, indicating that the effect on cation occlusion is indirect and not within the occlusion cavity. The ATP binding capacity remains unaltered by the modifications. Treatment of the K+-stabilized post-tryptic preparation of purified Na, K-ATPase revealed labeling of several cysteines by CPM, none of which were labeled with SDSM. Removal of K+ ions from the preparation, which we have previously shown is accompanied by release of the M5M6 hairpin to the supernatant (), causes changes in the organization of the C-terminal 21-kDa fragment. In particular Cys983 in M10 became labeled by both CPM and SDSM, pointing to a tight association between the C terminus and the M5M6 hairpin of the alpha-subunit.
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PMID:Identification of two conformationally sensitive cysteine residues at the extracellular surface of the Na,K-ATPase alpha-subunit. 903 May 96

Potassium transport across synaptosomes was studied under the influence of two synthetic pyrethroids, (Permethrin, without the cyano group) and Cypermethrin (CPM, with the cyano group). Synaptosomes were isolated from rat brain cerebral cortex and incubated with 40 microM of PM and CPM for 15 min at 37 degrees C. K+ release was monitored by a K(+)-sensitive electrode. CPM caused more K+ release from synaptosomes compared to PM. K+ transport is regulated by Na(+)-K(+)-ATPase, K(+)-ATPase and K+ channels. To understand the mode of action, synaptosomes were preincubated with 9.5 x 10(-3) M ouabain (inhibitor of Na(+)-K(+)-ATPase), 1.7 x 10(-2) M N-ethylmaleamide (K(+)-ATPase inhibitor), and 9.5 x 10(-5) M quinine sulfate (K(+)-channel blocker) for 15 min at 37 degrees C. In the presence of ouabain and N-ethylmaleamide, PM- and CPM-induced K+ release was decreased and in the presence of quinine sulfate, there was no release of K+. Furthermore, the studies indicated that PM and CPM significantly decreased K+ uptake.
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PMID:Modulation of K+ transport across synaptosomes of rat brain by synthetic pyrethroids. 910 17

Cys(10) is located in subdomain 1 of actin, which has an important role in the interaction of actin with myosin- and actin-binding proteins. Cys(10) was modified with fluorescence probes N-(iodoacetyl)N'-(5-sulfo-1-naphthyl)ethylene diamine (IAEDANS), 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM), or monobromo bimane (MBB) by the method of, J. Biol. Chem. 266:5508-5513). The specificity of Cys(10) modification was verified by showing that the 33-kDa subtilisin fragment of actin (residues 48-375), which contains all of the actin thiols but Cys(10), is not fluorescent. Cys(10) modification exposed a new site on actin to subtilisin cleavage. Edman degradation revealed this site to be between Ala(19) and Gly(20). The modification slightly increased the rate of epsilonATP-ATP exchange and decreased the rates of G-actin ATPase and polymerization. The activation of S1 ATPase by Cys(10)-modified F-actin showed small probe-dependent changes in the values of V(max) and K(M). The sliding speed of actin filaments in the in vitro motility assay remained unchanged upon modification of Cys(10). These results indicate that although the labeling of Cys(10) perturbs the structure of subdomain 1, the modified actin remains fully functional. The binding of S1 to actin filaments decreases the accessibility of Cys(10) probes to acrylamide and nitromethane quenchers. Because Cys(10) does not participate directly in either actin polymerization or S1 binding, our results indicate that actin-actin and actin-myosin interactions induce dynamic, allosteric changes in actin structure.
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PMID:Structural implications of the chemical modification of Cys(10) on actin. 1069 33

We introduced a reporting group into a critical position of the mitochondrial F(1)-ATPase in order to gain structural information about enzyme-ligand complexes. Incubation of 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM) with bovine heart mitochondrial F(1)-ATPase pretreated with 1 nM sodium arsenite modified the only cysteine residue in the gamma subunit (gamma-Cys(78)), resulting in an enzyme-CPM fluorescent complex (CPM-F(1)) with an ATPase activity similar to that of the native enzyme. Transferred fluorescence of F(1)-bound CPM occurred when different peptide fragments of naturally binding polypeptides carrying a pyrenylmaleimide (PM) moiety were bound to the enzyme. Fluorescence resonance energy transfer (RET) from PM bound to cysteine residues associated with Glu(40), Lys(47) and Lys(58) of fragments of the inhibitor protein (IF(1)) with CPM-F(1) occurred with an efficiency of approx. 20, 21 and 3% respectively. The distance at which the efficiency of energy transfer was 50%, R(0), for the CPM and PM donor/acceptor pair was 4.1 nm, indicating that the three IF(1) fragments must be within 6.7 nm of gamma-Cys(78). RET from the PM-bound hydrophilic fragment of c subunit (residues 37-42) of the F(1)F(0)-ATPase complex and CPM-bound gamma-Cys(78) occurred with an efficiency of approx. 30%, indicating a distance of 4.7 nm between the two fluorophores. Based on previous observations and on the present RET measurements, the hydrophilic loop of c subunit was located at the base of the F(1) foot, and the N-terminal region of IF(1) was located on the surface of F(1) in the lower part of the alpha(3)beta(3) hexamer ring.
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PMID:Fluorescence resonance energy transfer between coumarin-derived mitochondrial F(1)-ATPase gamma subunit and pyrenylmaleimide-labelled fragments of IF(1) and c subunit. 1182 53