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)

We have performed electron paramagnetic resonance (EPR) experiments on nitroxide spin labels incorporated into rabbit skeletal sarcoplasmic reticulum (SR), in order to investigate the physical and functional interactions between melittin, a small basic membrane-binding peptide, and the Ca-ATPase of SR. Melittin binding to SR substantially inhibits Ca(2+)-dependent ATPase activity at 25 degrees C, with half-maximal inhibition at 9 mol of melittin bound per mole of Ca-ATPase. Saturation transfer EPR (ST-EPR) of maleimide spin-labeled Ca-ATPase showed that melittin decreases the submillisecond rotational mobility of the enzyme, with a 4-fold increase in the effective rotational correlation time (tau r) at a melittin/Ca-ATPase mole ratio of 10:1. This decreased rotational motion is consistent with melittin-induced aggregation of the Ca-ATPase. Conventional EPR was used to measure the submicrosecond rotational dynamics of spin-labeled stearic acid probes incorporated into SR. Melittin binding to SR at a melittin/Ca-ATPase mole ratio of 10:1 decreases lipid hydrocarbon chain mobility (fluidity) 25% near the surface of the membrane, but only 5% near the center of the bilayer. This gradient effect of melittin on SR fluidity suggests that melittin interacts primarily with the membrane surface. For all of these melittin effects (on enzymatic activity, protein mobility, and fluidity), increasing the ionic strength lessened the effect of melittin but did not alleviate it entirely. This is consistent with a melittin-SR interaction characterized by both hydrophobic and electrostatic forces. Since the effect of melittin on lipid fluidity alone is too small to account for the large inhibition of Ca-ATPase rotational mobility and enzymatic activity, we propose that melittin inhibits the ATPase primarily through its capacity to aggregate the enzyme, consistent with previous observations of decreased Ca-ATPase activity under conditions that decrease protein rotational mobility.
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PMID:Effects of melittin on molecular dynamics and Ca-ATPase activity in sarcoplasmic reticulum membranes: electron paramagnetic resonance. 164 24

We have studied the effect of melittin, a basic membrane-binding peptide, on Ca-ATPase activity and on protein and lipid dynamics in skeletal sarcoplasmic reticulum (SR), using time-resolved phosphorescence and fluorescence spectroscopy. Melittin completely inhibits Ca-ATPase activity, with half-maximal inhibition at 9 +/- 1 mol of melittin bound to the membrane per mole of ATPase (0.1 mol of melittin per mole of lipid). The time-resolved phosphorescence anisotropy (TPA) decay of the Ca-ATPase labeled with erythrosin isothiocyanate (ERITC) shows that melittin restricts microsecond protein rotational motion. At 25 degrees C in the absence of melittin, the TPA is characterized by three decay components, corresponding to a rapid segmental motion (correlation time phi 1 = 2-3 microseconds), the uniaxial rotation of monomers or dimers (phi 2 = 16-22 microseconds), and the uniaxial rotation of larger oligomers (phi 3 = 90-140 microseconds). The effect of melittin is primarily to decrease the fraction of the more mobile monomer/dimer species (A2) while increasing the fractions of the larger oligomer (A3) and very large aggregates (A infinity). Time-resolved fluorescence anisotropy of the lipid-soluble probe diphenylhexatriene (DPH) shows only a slight increase in the lipid hydrocarbon chain effective order parameter, corresponding to an increase in lipid viscosity that is too small to account for the large decrease in protein mobility or inhibition of Ca-ATPase activity. Thus the inhibitory effect of melittin correlates with its capacity to aggregate the Ca-ATPase and is consistent with previously reported inhibition of this enzyme under conditions that increase protein-protein interactions.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Effects of melittin on molecular dynamics and Ca-ATPase activity in sarcoplasmic reticulum membranes: time-resolved optical anisotropy. 164 30

Plasma membranes were prepared from red beet (Beta vulgaris L.) storage tissue by partition in an aqueous two-phase system. A highly active proton-translocating ATPase was purified from these membranes by lysophosphatidylcholine extraction and glycerol density gradient centrifugation. The ATPase activity was inhibited by vanadate or dicyclohexyl carbodiimide, but was insensitive to azide, nitrate and molybdate at concentrations which inhibit the F1ATPase, the tonoplast ATPase, and acid phosphatase. Inhibition by vanadate was consistent with a non-competitive mechanism, with Ki = 10 microM. The Km for Mg-ATP was about 1 mM, magnesium ions were required, and the activity was stimulated by KCl and by lysophosphatidylcholine. The optimal pH was 6.5. The molecular mass by gel filtration in the presence of 2 g/liter octyl glucoside was 600 kDa, while dodecyl sulfate gel electrophoresis gave a polypeptide molecular mass of 100 kDa. After blotting onto nitrocellulose, the purified enzyme did not bind concanavalin A, although a concanavalin A-binding peptide of the plasma membrane runs to nearly the same position on the gel and showed some tendency to co-purify with the ATPase. Phospholipid vesicles into which the purified ATPase had been incorporated by the freeze-thaw technique showed vanadate-sensitive, ATP-dependent proton uptake. When the ATPase was reconstituted into lipid membranes at high protein to lipid ratios and incubated with ATP, two-dimensionally crystalline arrays of protein molecules were formed.
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PMID:Purification and characterization of the proton translocating plasma membrane ATPase of red beet storage tissue. 214 2

We have previously shown that melittin, a bee venom peptide, potently inhibited the catalytic and transport functions of rabbit gastric (H+ + K+)ATPase. A radioactive photoaffinity analog of melittin, ([125I]azidosalicylyl melittin), labeled the (H+ + K+)ATPase. These results suggested that melittin exerted inhibitory effects through direct interaction with the (H+ + K+)ATPase. In this study we attempt to define the melittin-binding domain of the (H+ + K+)ATPase using conformation-dependent proteolytic fragmentation of [125I]azidosalicylyl melittin-labeled hog gastric (H+ + K+)ATPase. In the presence of KCl (E2 form) the 95,000-Da [125I]-azidosalicylyl melittin-labeled (H+ + K+)ATPase was cleaved by trypsin to a 40,000-Da NH2-terminal tryptic fragment and a 56,000-Da COOH-terminal fragment through cleavage at Arg 454 of the (H+ + K+)ATPase. The 40,000-Da fragment was labeled by [125I]-azidosalicylyl melittin. The 56,000-Da fragment was not labeled. When unmodified (H+ + K+)ATPase was trypsinized in the presence of KCl, and the fragments were then reacted with [125I]azidosalicylyl melittin, similar tryptic fragmentation results were obtained. In the absence of KCl (E1 form), the 56,000- and 40,000-Da fragments did not accumulate. Chymotryptic hydrolysis of [125I]azidosalicylyl melittin-labeled (H+ + K+)-ATPase was very slow in the presence of KCl (E2 form). In the absence of KCl (E1 form), chymotryptic hydrolysis was more rapid, with accumulation of a major 42,000-Da fragment which was radiolabeled. The melittin-binding region on the (H+ + K+)ATPase is N-terminal to Arg 454 of the (H+ + K+)ATPase. This region is known to contain the aspartyl phosphate residue (Asp 385), the site of phosphoenzyme formation on the (H+ + K+)ATPase. Melittin is also known to bind to calmodulin and other proteins. Another known calmodulin-binding peptide with a different sequence but similar structure, Trp-3, (Leu-Lys-Trp-Lys-Lys-Leu-Leu-Lys-Leu-Leu-Lys-Lys-Leu-Leu-Lys-Leu-Gly) also inhibited the (H+ + K+)ATPase and label incorporation by [125I]azidosalicylyl melittin. These Trp-3 results suggested that the (H+ + K+)ATPase contains a peptide-binding domain which is similar to the peptide-binding domains found on other melittin-binding proteins.
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PMID:[125I]azidosalicylyl melittin binding domains: evidence for a polypeptide receptor on the gastric (H+ + K+)ATPase. 215 80

Higher plant cells have one or more vacuoles important for maintaining cell turgor and for the transport and storage of ions and metabolites. One driving force for solute transport across the vacuolar membrane (tonoplast) is provided by an ATP-dependent electrogenic H+ pump. The tonoplast H+-pumping ATPase from oat roots has been solubilized with Triton X-100 and purified 16-fold by Sepharose 4B chromatography. The partially purified enzyme was sensitive to the same inhibitors (N-ethylmaleimide, N,N'-dicyclohexylcarbodiimide (DCCD), 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid, and NO-3) as the native membrane-bound enzyme. The partially purified enzyme was stimulated by Cl- (Km(app) = 1.0 mM) and hydrolyzed ATP with a Km(app) of 0.25 mM. Thus, the partially purified tonoplast ATPase has retained the properties of the native membrane-bound enzyme. [14C]DCCD labeled a single polypeptide (14-18 kDa) in the purified tonoplast ATPase preparation. Two major polypeptides, 72 and 60 kDa, that copurified with the ATPase activity and the 14-18-kDa DCCD-binding peptide are postulated to be subunits of a holoenzyme of 300-600 kDa (estimated by gel filtration). Despite several catalytic similarities with the mitochondrial H+-ATPase, the major polypeptides of the tonoplast ATPase differed in mass from the alpha and beta subunits (58 and 55 kDa) and the [14C] DCCD-binding proteolipid (8 kDa) of the oat F1F0-ATPase.
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PMID:Properties of the partially purified tonoplast H+-pumping ATPase from oat roots. 286 99

When scallop S1(+LC) (formerly called CaMg S1) is digested by trypsin, the heavy chain degrades while the two light chains remain complexed to each other and a peptide fragment of the heavy chain. The three components of the complex comigrate during electrophoresis under nondissociating conditions and can be purified by chromatography and concentrated by precipitation with ammonium sulphate in the presence of millimolar calcium ions. The truncated regulatory light chain remains associated with the binary complex consisting of the peptide and essential light chain as long as divalent cations are present; in the presence of EDTA it dissociates. This behaviour of the light chains-peptide complex mimics that of the intact molecule. The effect of bound light chains and bound actin on the susceptibility to tryptic digestion was studied using scallop S1(+LC) and S1(-LC) (EDTA S1 according to previous nomenclature). The heavy chains of both types of S1 are labile and have two main sites susceptible to proteolysis. Tryptic digestion on site A produces an N-terminal peptide of around 70 000 and a C-terminal 24 000 fragment from S1(+LC) and a 20 000 C-terminal fragment from S1(-LC); the latter is prone to further proteolysis. Thus S1(-LC), produced in the absence of bound regulatory light chain is shorter on the C-terminal end. Proteolysis on site A abolishes actin-activated ATPase activity; the latter is prevented by digesting acto-S1. The rate of tryptic digestion on site B is somewhat slower than on site A; when either S1 is split at this site an N-terminal 63 000 peptide is produced. The corresponding C-terminal peptide can be obtained from acto-S1 when hydrolysis on site A is prevented; this is estimated as around 31 000 derived from S1(+LC) and 28 000 derived from S1(-LC). The results are compared with similar experiments where vertebrate subfragments were digested by trypsin and the possible localization of the light-chain binding peptide in the intact heavy chain is discussed.
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PMID:Tryptic digestion of scallop S1: evidence for a complex between the two light-chains and a heavy-chain peptide. 623 96

The conditions of nucleotide binding to native, though partly purified, Ca(2+)-ATPase from SR as well as the stoichiometry of nucleotide and strontium binding and the phosphorylation capacity was reevaluated. Binding of MgADP appeared to be aberrant whereas even high-affinity binding of [14C]-ADP took place in the absence of Mg2+. Also low-affinity ATP binding was possible in the absence of divalent cations. A heterogeneity in ADP binding compatible with a two-component model in the absence of thapsigargin was changed to an apparent homogeneity of low-affinity receptors following a mole:mole interaction of enzyme and thapsigargin. Since the affinity of both components was reduced by thapsigargin, high- as well as low-affinity ADP binding seem to be specific and probably to the substrate receptor proper. Analysis of ADP binding isotherms in the absence of Mg2+ according to a model of two independent populations of sites was compatible with a binding capacity of 8.49 +/- 0.43 nmoles/mg protein corresponding to a molecular mass of 118 +/- 6 kD per ADP site. The same total binding capacity was found for ATP. The phosphorylation capacity corresponded to more than one and less than two approximately P per two 110-kD peptides (formally one approximately P per 154 kD protein). Specific binding of Ca2+ and the congener Sr2+ to SR Ca(2+)-ATPase was compatible with their interaction with a single population of sites. The binding capacity was equal to one divalent cation per nucleotide binding peptide. The binding of one nucleotide and one divalent cation per approximately 110 kD peptide and the absence of cooperativity in divalent cation binding might imply that Ca(2+)-ATPase works as a monomer.
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PMID:Binding of ADP to sarcoplasmic reticulum Ca(2+)-ATPase in the absence of Mg2+ is specifically inhibited by thapsigargin: observations on the ligand stoichiometry. 874 53

The catalytic domain of myosin light chain kinase (MLCK) not only exerts kinase activity to phosphorylate the 20 kDa light chain but also inhibits the actin-myosin interaction. The site of action of this novel role of the domain has been suggested to be myosin [Okagaki et al. (1999) J. Biochem. 125, 619-626]. In this study, we have analyzed the amino acid sequences of MLCK and myosin that are involved in the inhibition. The ATP-binding peptide of Gly526-Lys548 of chicken gizzard MLCK exerted the inhibitory effect on the movement of actin filaments on a myosin-coated glass surface. However, the peptide that neighbors the sequence failed to inhibit the movement. The inhibition of the ATP-binding peptide was confirmed by measuring ATPase activities of the myosin. The inhibition by parent MLCK of the movement was relieved by the 20 kDa light chain, but not by the 17 kDa myosin light chain. The peptide of the 20 kDa light chain sequence of Ser1-Glu29 also relieved the inhibition. Thus, the interaction of the ATP-binding sequence with the 20 kDa light chain sequence should cause the inhibition of the actin-myosin interaction. Concerning the regulation of the inhibition, calmodulin relieved the inhibitory effect of MLCK on the movement of actin filaments. The calmodulin-binding peptide (Ala796 Ser815) prevented the relief, suggesting the involvement of this sequence. Thus, the mode of regulation by Ca2+ and calmodulin of the novel role of the catalytic domain is similar, but not identical, to the mode of regulation of the kinase activity of the domain.
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PMID:Inhibitory effect of the catalytic domain of myosin light chain kinase on actin-myosin interaction: insight into the mode of inhibition. 1034 7

Calmodulin (CaM) and Ca(2+)/CaM-dependent protein kinase II (CaM kinase) are tightly associated with cardiac sarcoplasmic reticulum (SR) and are implicated in the regulation of transmembrane Ca(2+) cycling. In order to assess the importance of membrane-associated CaM in modulating the Ca(2+) pump (Ca(2+)-ATPase) function of SR, the present study investigated the effects of a synthetic, high affinity CaM-binding peptide (CaM BP; amino acid sequence, LKWKKLLKLLKKLLKLG) on the ATP-energized Ca(2+) uptake, Ca(2+)-stimulated ATP hydrolysis, and CaM kinase-mediated protein phosphorylation in rabbit cardiac SR vesicles. The results revealed a strong concentration-dependent inhibitory action of CaM BP on Ca(2+) uptake and Ca(2+)-ATPase activities of SR (50% inhibition at approximately 2-3 microM CaM BP). The inhibition, which followed the association of CaM BP with its SR target(s), was of rapid onset (manifested within 30 s) and was accompanied by a decrease in V(max) of Ca(2+) uptake, unaltered K(0.5) for Ca(2+) activation of Ca(2+) transport, and a 10-fold decrease in the apparent affinity of the Ca(2+)-ATPase for its substrate, ATP. Thus, the mechanism of inhibition involved alterations at the catalytic site but not the Ca(2+)-binding sites of the Ca(2+)-ATPase. Endogenous CaM kinase-mediated phosphorylation of Ca(2+)-ATPase, phospholamban, and ryanodine receptor-Ca(2+) release channel was also strongly inhibited by CaM BP. The inhibitory action of CaM BP on SR Ca(2+) pump function and protein phosphorylation was fully reversed by exogenous CaM (1-3 microM). A peptide inhibitor of CaM kinase markedly attenuated the ability of CaM to reverse CaM BP-mediated inhibition of Ca(2+) transport. These findings suggest a critical role for membrane-bound CaM in controlling the velocity of Ca(2+) pumping in native cardiac SR. Consistent with its ability to inhibit SR Ca(2+) pump function, CaM BP (1-2.5 microM) caused marked depression of contractility and diastolic dysfunction in isolated perfused, spontaneously beating rabbit heart preparations. Full or partial recovery of contractile function occurred gradually following withdrawal of CaM BP from the perfusate, presumably due to slow dissociation of CaM BP from its target sites promoted by endogenous cytosolic CaM.
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PMID:Reversible inhibition of the calcium-pumping ATPase in native cardiac sarcoplasmic reticulum by a calmodulin-binding peptide. Evidence for calmodulin-dependent regulation of the V(max) of calcium transport. 1066 Jun 12

The effects of a number of phenothiazines and other calmodulin antagonists on the Ca(2+)-ATPase activity of sarcoplasmic reticulum (SR) and endoplasmic reticulum (ER) were investigated. The drugs used in this study were trifluoperazine, calmidazolium, fluphenazine, chlorpromazine, W-7, and calmodulin-binding peptide. Our results showed that calmidazolium and calmodulin-binding peptide were the most potent inhibitors of skeletal muscle SR Ca(2+)-ATPase activity (isoform SERCA 1) (IC(50) values of 0.5 and 7 microM, respectively), while W-7 was the least potent inhibitor (IC(50), 125 microM). All of the antagonists had little effect on the cerebellar ER Ca(2+)-ATPase activity (isoform SERCA 2b), except for trifluoperazine, which had a biphasic effect, causing stimulation at low concentrations and inhibition at higher concentrations. Our results suggest that the effects of these calmodulin antagonists are independent of calmodulin and that they inhibit the Ca(2+)-ATPase in an isoform-specific manner. It was found that these antagonists inhibit the skeletal muscle isoform of the Ca(2+) pump by altering the Ca(2+) affinity and the associated Ca(2+)-binding steps, as well as possibly stabilising the E1 conformational state of the enzyme.
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PMID:The effects of phenothiazines and other calmodulin antagonists on the sarcoplasmic and endoplasmic reticulum Ca(2+) pumps. 1110 94


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