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)

Previous studies had led to the conclusion that the globular, single-headed myosins IA and IB from Acanthamoeba castellanii contain two actin-binding sites: one associated with the catalytic site and whose binding to F-actin activates the Mg2+-ATPase activity and a second site whose binding results in the cross-linking of actin filaments and makes the actin-activated ATPase activity positively cooperative with respect to myosin I concentration. We have now prepared a 100,000-Da NH2-terminal peptide and a 30,000-Da COOH-terminal peptide by alpha-chymotryptic digestion of the myosin IA heavy chain. The intact 17,000-Da light chain remained associated with the 100,000-Da fragment, which also contained the serine residue that must be phosphorylated for expression of actin-activated ATPase activity by native myosin IA. The 30,000-Da peptide, which contained 34% glycine and 21% proline, bound to F-actin with a KD less than 0.5 microM in the presence or absence of ATP but had no ATPase activity. The 100,000-Da peptide bound to F-actin with KD = 0.4-0.8 microM in the presence of 2 mM MgATP and KD less than 0.01 microM in the absence of MgATP. In contrast to native myosin IA, neither peptide cross-linked actin filaments. The phosphorylated 100,000-Da peptide had actin-activated ATPase activity with the same Vmax as that of native phosphorylated myosin IA but this activity displayed simple, noncooperative hyperbolic dependence on the actin concentration in contrast to the complex cooperative kinetics observed with native myosin IA. These results provide direct experimental evidence for the presence of two actin-binding sites on myosin IA, as was suggested by enzyme kinetic and filament cross-linking data, and also for the previously proposed mechanism by which monomeric myosins I could support contractile activities.
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PMID:ATPase activities and actin-binding properties of subfragments of Acanthamoeba myosin IA. 294 92

The erythrocyte plasma membrane Ca2+-pumping ATPase is known to form an acyl-phosphate catalytic intermediate, but there is otherwise little structural information linking it to the other mammalian ion-pumping ATPases which also form phosphorylated intermediates (the Na+, K+-ATPase of plasma membranes, the Ca2+-ATPase of sarcoplasmic reticulum, and the H+, K+-ATPase of gastric mucosa). We show here that this enzyme possesses a fluorescein isothiocyanate-reactive region similar to that possessed by these other ATPases. Low concentrations (10 microM) of fluorescein isothiocyanate inhibit the ATPase activity of this pump, and this inhibition is prevented by 4 mM ATP. ATP also inhibits the reaction of fluorescein isothiocyanate with a single amino acid residue on the 138-kDa polypeptide chain. A tryptic fragment containing the fluorescein-conjugated residue was isolated by high pressure liquid chromatography. The sequence of this peptide was determined to be NH2-Met1-Tyr2-Ser3-Lys4-Gly5-Ala6-Ser7-Glu8++ +-Ile9-Ile10-Leu11-Arg12-COOH; fluorescein isothiocyanate reacts with the lysine residue. The identities of residues 4-8 are the same as those in a sequence common to the other ATPases mentioned above, except that serine-7 of this sequence is changed to a proline in those ATPases. This substitution, sometimes not considered a homologous one, is not expected to have a major effect on the secondary structure or polarity of this region. Outside of this 5-residue core region of the fluorescein isothiocyanate-reactive site, the homologies among the different ion-pumping ATPases are limited.
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PMID:The ATP-binding site of the erythrocyte membrane Ca2+ pump. Amino acid sequence of the fluorescein isothiocyanate-reactive region. 295 52

The actin-dependent ATPase activity of myosin is retained in the separated heads (S1) which contain the NH2-terminal 95-kDa heavy chain fragment and one or two light chains. The S1 heavy chain can be degraded further by limited trypsin treatment into characteristic 25-, 50-, and 20-kDa peptides, in this order from the NH2-terminal end. The 20-kDa peptide contains an actin-binding site and SH1 and SH2, two thiols whose modification dramatically affects ATPase activity. By treating myosin filaments with trypsin at 4 degrees C in the presence of 2 mM MgCl2, we have now obtained preferential cleavage at the 50-20-kDa heavy chain site without any cleavage at the head-rod junction and hinge region in the rod. Incubation of these trypsinized filaments at 37 degrees C in the presence of MgATP released a new S1 fraction which lacked the COOH-terminal 20-kDa heavy chain peptide region. This fraction, termed S1'(75K), has more than 50% of the actin-activated Mg2+-ATPase activity of S1 and the characteristic Ca2+-ATPase and K+-EDTA ATPase activities of myosin. These results show that SH1 and SH2 are not essential for ATPase activity and that binding of actin to the 20-kDa region is not essential for the enhancement of the Mg2+-ATPase activity.
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PMID:A new, smaller actin-activatable myosin subfragment 1 which lacks the 20-kDa, SH1 and SH2 peptide. 295 48

Acanthamoeba myosin IA is a globular protein composed of a 140-kDa heavy chain and a 17-kDa light chain. It expresses high actin-activated Mg2+-ATPase activity when one serine on the heavy chain is phosphorylated. We previously showed that chymotrypsin cleaves the heavy chain into a COOH-terminal 27-kDa peptide that can bind to F-actin but has no ATPase activity and a complex containing the NH2-terminal 112-kDa peptide and the light chain. The complex also binds F-actin and has full actin-activated Mg2+-ATPase activity when the regulatory site is phosphorylated. We have now localized the ATP binding site to within 27 kDa of the NH2 terminus and the regulatory phosphorylatable serine to a 20-kDa region between 38 and 58 kDa of the NH2 terminus. Under controlled conditions, trypsin cleaves the heavy chain at two sites, 38 and 112 kDa from the NH2 terminus, producing a COOH-terminal 27-kDa peptide similar to that produced by chymotrypsin and a complex consisting of an NH2-terminal kDa peptide, a central 74-kDa peptide, and the light chain. This complex is similar to the chymotryptic complex but for the cleavage which separates the 38- and 74-kDa peptides. The tryptic complex has full (K+, EDTA)-ATPase activity (the catalytic site is functional) and normal ATP-sensitive actin-binding properties. However, the actin-activated Mg2+-ATPase activity and the F-actin-binding characteristics of the tryptic complex are no longer sensitive to phosphorylation of the regulatory serine. Therefore, cleavage between the phosphorylation site and the ATP-binding site inhibits the effects of phosphorylation on actin binding and actin-activated Mg2+-ATPase activity without abolishing the interactions between the ATP- and actin-binding sites.
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PMID:Limited tryptic digestion of Acanthamoeba myosin IA abolishes regulation of actin-activated ATPase activity by heavy chain phosphorylation. 295 54

Acanthamoeba myosin IB contains a 125-kDa heavy chain that has high actin-activated Mg2+-ATPase activity when 1 serine residue is phosphorylated. The heavy chain contains two F-actin-binding sites, one associated with the catalytic site and a second which allows myosin IB to cross-link actin filaments but has no direct effect on catalytic activity. Tryptic digestion of the heavy chain initially produces an NH2-terminal 62-kDa peptide that contains the ATP-binding site and the regulatory phosphorylation site, and a COOH-terminal 68-kDa peptide. F-actin, in the absence of ATP, protects this site and tryptic cleavage then produces an NH2-terminal 80-kDa peptide. Both the 62- and the 80-kDa peptides retain the (NH+4,EDTA)-ATPase activity of native myosin IB and both bind to F-actin in an ATP-sensitive manner. However, only the 80-kDa peptide retains a major portion of the actin-activated Mg2+-ATPase activity. This activity requires phosphorylation of the 80-kDa peptide by myosin I heavy chain kinase but, in contrast to the activity of intact myosin IB, it has a simple, hyperbolic dependence on the concentration of F-actin. Also unlike myosin IB, the 80-kDa peptide cannot cross-link F-actin filaments indicating the presence of only a single actin-binding site. These results allow the assignment of the actin-binding site involved in catalytic activity to the region near, and possibly on both sides of, the tryptic cleavage site 62 kDa from the NH2 terminus, and the second actin-binding site to the COOH-terminal 45-kDa domain. Thus, the NH2-terminal 80 kDa of the myosin IB heavy chain is functionally similar to the 93-kDa subfragment 1 of muscle myosin and most likely has a similar organization of functional domains.
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PMID:Localization of the actin-binding sites of Acanthamoeba myosin IB and effect of limited proteolysis on its actin-activated Mg2+-ATPase activity. 296 46

A Ca++-dependent secretion of norepinephrine ([3H]NE) was evoked in adrenergic nerve endings in rat heart ventricle slices incubated in a modified Krebs-HCO3 medium containing choline Cl as the replacement for NaCl (Ch+-Ca++). Exogenous ATP inhibited secretion and lithium ion, a known inhibitor of NE uptake dependent upon Mg++-ATPase activity in vesicles (but not ouabain) prevented the response to ATP (Bogdanski, 1983,1986). It was suggested that vesicles attached to the axolemma recaptured [3H]NE from the extracellular fluid. This report indicates that other known inhibitors of uptake in isolated vesicles also inhibited the response to ATP in the attached vesicles. Included were two inhibitors of Mg++-ATPase activity, N-ethylmaleimide (NEM) and dicyclohexylcarbodiimide (DCCD), and the proton transporters 2,4-dinitrophenol (2,4-DNP 1.0 mM) and chlorpromazine (CPZ). Potassium ionophores (valinomycin with 2,4-DNP 0.1 mM, and nigericin) and a pH neutralizing reagent for vesicles (NH3 from ammonium sulfate in solution) were also effective. The uptake inhibitors, except 2,4-DNP, could also increase the rate of depletion of stored NE and its deamination in nonsecreting nerve endings incubated in Krebs-HCO3 (KRB) medium. Valinomycin by itself stimulated uptake in the presence of ATP. It is suggested that mechanisms of uptake and retention of NE in isolated vesicles (symposium (1982) Fed. Proc. 41:2742-2780) apply to the axoplasmic vesicles as well. Thus, the activity of Mg++-ATPase drives proton transport to establish the electrochemical gradients of H+, which drive the transport of NE. A lowering of the gradients can mobilize amines and evoke secretion.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Norepinephrine uptake dependent upon apparent Mg++-ATPase activity and proton transport in storage vesicles in axoplasm. 297 43

Ultraviolet irradiation above 300 nm of the stable MgADP-orthovanadate (Vi)-myosin subfragment 1 (S1) complex resulted in covalent modification of the S1 and in the rapid release of trapped MgADP and Vi. This photomodified S1 had Ca2+ATPase activity 4-5-fold higher than that of the non-irradiated control S1, while the K+EDTA-ATPase activity was below 10% of controls. There was a linear correlation between the activation of the Ca2+ATPase and the release of both ADP and Vi with irradiation time. Analysis of the total number of thiols and the ability of photomodified S1 to retrap MgADP by cross-linking SH1 and SH2 with various bifunctional thiol reagents indicated that the photomodification did not involve these reactive thiols. Irradiation of the S1-MgADP-Vi complex caused a large increase in absorbance of the enzyme at 270 nm which was correlated with the release of Vi from the active site, suggesting an aromatic amino acid(s) was (were) involved. However, analysis by three different methods showed no loss of tryptophan. All the irradiation-dependent phenomena could be prevented by replacing Mg2+ with either Co2+, Mn2+, or Ni2+. Unlike previous irradiation studies of Vi-dynein complexes [Lee-Eiford, A., Ow, R. A., & Gibbons, I. R. (1986) J. Biol. Chem. 261, 2337-2342], no peptide bonds were cleaved in photomodified S1. Photomodified S1 was able to retrap MgADP-Vi at levels similar to unmodified S1. Upon irradiation of the photomodified S1-MgADP-Vi complex, MgADP and Vi were again released from the active site, resulting in heavy chain cleavage to form NH2-terminal 21-kDa and COOH-terminal 74-kDa peptides. All evidence indicates that this new photomodification and subsequent chain cleavage occur specifically at the active site.
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PMID:UV-induced vanadate-dependent modification and cleavage of skeletal myosin subfragment 1 heavy chain. 1. Evidence for active site modification. 297 86

The sequences of the first 14 amino acids of the (Na+,K+)-ATPase catalytic subunits from rat kidney (alpha) and rat brain axolemma (alpha(+)) have been determined. They are: (alpha), NH2-Gly-Arg-Asp-Lys-Tyr-Glu-Pro-Ala-Ala-Val-Ser-Glu-His-Gly; (alpha(+)), NH2-Gly-Arg-Glu-Tyr-Ser-Pro-Ala-Ala-Glu-Val-Ala-Glu-Val-Gly. Although they are highly homologous, it is clear these sequences are also sufficiently different to conclude they are the products of different genes, or at least different exons of the same, differentially spliced, gene. Among mammals, the amino terminal sequence of the kidney alpha chain is essentially invariant. Thus this section of the (Na+,K+)-ATPase molecule is more highly conserved in one tissue between several species than between different tissues in the same species. This may reflect upon the difference in function of the alpha and alpha(+) isozymes of (Na+,K+)-ATPase.
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PMID:The catalytic subunits of the (Na+,K+)-ATPase alpha and alpha(+) isozymes are the products of different genes. 299 84

Purified dog kidney (Na+,K+)-ATPase was reacted with tritiated sodium borohydride after treatment with neuraminidase and galactose oxidase. This procedure did not affect the ATPase activity of the enzyme, and all of the covalently bound radioactivity was found in the beta subunit (Mr 54 000). Papain digestion of the tritiated enzyme produced two labeled fragments of Mr 40 000 and 16 000. Further proteolysis generated an Mr 31 000 peptide from the larger fragment. Unlike the tryptic and chymotryptic sites of the alpha subunit, the sites of papain hydrolysis were insensitive to conformations of the (Na+,K+)-ATPase. Determination of the NH2-terminal sequences was used to arrange the fragments within the linear map of the beta chain. Finally, none of the labeled peptides was released from the membrane under nondenaturing conditions. These results are consistent with a model of the beta subunit containing a 40 000-dalton NH2-terminal piece and a 16 000-dalton COOH-terminal piece. Both fragments have extracellularly exposed carbohydrate and at least one membrane-bound domain.
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PMID:Papain fragmentation of the (Na+,K+)-ATPase beta subunit reveals multiple membrane-bound domains. 300 27

Three methods were used to assess protein concentration in membrane-bound Na,K-ATPase preparations: standard Lowry assay, Kjeldahl nitrogen determination and amino acid analysis. While the first two methods showed excellent agreement, the third one always gave a lower value which varied drastically depending on the condition of sample treatment before amino acid analysis. This result reinforces the Lowry method in assessing the true concentration of Na,K-ATPase protein and suggests 250 kDa to be a true estimate of the molecular mass of the smallest ligand-binding unit of the enzyme. The cyanate method reveals two NH2-terminal residues of the beta-subunit (NH2-Ala) and one such residue of the alpha-subunit (NH2-Gly) per ligand-binding unit. From the data on equimolarity of the alpha- and beta-subunits in Na,K-ATPase this suggests that the enzyme molecule is composed of two alpha beta-protomers, one possessing a modified (presumably an N-blocked) alpha-subunit.
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PMID:Evidence for a diprotomeric structure of Na,K-ATPase. Accurate determination of protein concentration and quantitative end-group analysis. 300 59


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