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)

Protease Ti, a new ATP-dependent protease in Escherichia coli, degrades proteins and ATP in a linked process, but these two hydrolytic functions are catalyzed by distinct components of the enzyme. To clarify the enzyme's specificity and the role of ATP, a variety of fluorogenic peptides were tested as possible substrates for protease Ti or its two components. Protease Ti rapidly hydrolyzed N-succinyl(Suc)-Leu-Tyr-amidomethylcoumarin (AMC) (Km = 1.3 mM) which is not degraded by protease La, the other ATP-dependent protease in E. coli. Protease Ti also hydrolyzed, but slowly, Suc-Ala-Ala-Phe-AMC and Suc-Leu-Leu-Val-Tyr-AMC. However, it showed little or no activity against basic or other hydrophobic peptides, including ones degraded rapidly by protease La. Component P, which contains the serine-active site, by itself rapidly degrades the same peptides as the intact enzyme. Addition of component A, which contains the ATP-hydrolyzing site and is necessary for protein degradation, had little or no effect on peptide hydrolysis. N-Ethylmaleimide, which inactivates the ATPase, did not inhibit peptide hydrolysis. In addition, this peptide did not stimulate the ATPase activity of component A (unlike protein substrates). Thus, although the serine-active site on component P is unable to degrade proteins, it is fully functional against small peptides in the absence of ATP. At high concentrations, Suc-Leu-Tyr-AMC caused a complete inhibition of casein breakdown, and diisopropylfluorophosphate blocked similarly the hydrolysis of both protein and peptide substrates. Thus, both substrates seem to be hydrolyzed at the same active site on component P, and ATP hydrolysis by component A either unmasks or enlarges this proteolytic site such that large proteins can gain access to it.
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PMID:Protease Ti from Escherichia coli requires ATP hydrolysis for protein breakdown but not for hydrolysis of small peptides. 264 53

Several newly synthesized 4-hydroxycinnamamide derivatives such as 3-(3',5'-di-isopropyl-4'-hydroxybenzylidene)-2-oxindol (ST 280), 3-(3',5'-di-methylthiomethyl-4'-hydroxybenzylidene)-2-oxindole (ST 458), alpha-cyano-3-ethoxy-4-hydroxy-5-phenylthiomethylcinnamamide (ST 638) and 3-(3'-ethoxy-4'-hydroxy-5'-phenylthiomethylbenzylidene)-2-pyrol idinone (ST 642) were found to inhibit tyrosine-specific protein kinase activity of the epidermal growth factor (EGF) receptor with IC50 values of 0.44 microM, 0.44 microM, 0.37 microM and 0.85 microM, respectively. None of them showed inhibitory effect on the enzyme activities of serine- and/or threonine-specific protein kinases such as cAMP-dependent protein kinase, Ca2+/phospholipid-dependent protein kinase C, casein kinase I and casein kinase II. In addition, none of them had effect on Na+/K+-ATPase or 5'-nucleotidase. The results suggest that the compound ST 280, ST 458, ST 638 and ST 642 are potent and specific inhibitors of tyrosine-specific protein kinase.
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PMID:Specific inhibitors of tyrosine-specific protein kinase, synthetic 4-hydroxycinnamamide derivatives. 282 Mar 97

The effect of phosphorylation on the ability of simian virus 40 large T antigen to stimulate DNA synthesis in vitro was tested. Treatment of affinity-purified large T antigen with calf intestinal alkaline phosphatase resulted in the removal of 70 to 80% of the phosphate residues. Only serine-bound phosphate residues were affected. Phosphatase-treated large T antigen stimulated in vitro DNA synthesis fourfold over the untreated control. The stimulation was strongest at early times of DNA replication. At later times, DNA replication proceeded at equal rates with dephosphorylated and untreated large T antigen. The ATPase activity of large T antigen was not affected by phosphatase treatment. The origin-binding activity of large T antigen was tested over a wide range of large T antigen to DNA ratios, including DNA excess, and in the presence and absence of carrier DNA. Under no condition was an effect of dephosphorylation of large T antigen on its DNA-binding activity observed. These findings might indicate that phosphorylation at serine residues modulates the interaction of large T antigen with cellular factors. During DNA synthesis large T antigen was substantially rephosphorylated by kinases in the HeLa cell extract. As shown by two-dimensional peptide mapping, this phosphorylation occurred at all known in vivo sites. No phosphatase and protease activities were detectable in the HeLa cell extract.
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PMID:Removal of serine phosphates from simian virus 40 large T antigen increases its ability to stimulate DNA replication in vitro but has no effect on ATPase and DNA binding. 282 47

Plasma from insulin-dependent diabetics shows an increased ability to specifically activate the (Na-K)ATPase from different sources. Several protease inhibitors like phenyl methyl sulfonyl fluoride, trypsin inhibitor, antithrombin III and aprotinin, produced a significant dose-dependent inhibition of the stimulatory effect produced by a 1/100 final dilution of plasma on the beef heart (Na-K)ATPase activity. Serine proteases employed at scalar concentrations in the ATPase medium gave a dose-dependent stimulation of the enzyme activity as did diabetic plasma. The maximum percent stimulation of the (Na-K)ATPase activity (about 60%) was reached by 0.56 microgram/ml of thrombin, 0.50 microgram/ml of kallikrein and 0.55 microgram/ml of trypsin. The protease-induced ATPase stimulation was significantly reduced by antithrombin III, trypsin inhibitor and by aprotinin. A partial purification of the activating plasma factor was obtained by eluting plasma on a heparin-Sepharose column. Two (Na-K)ATPase stimulating fractions were found, which eluted with 1.0 and 3.0 mol/l NaCl, respectively. Half-maximal stimulation of the enzyme occurred with 3.4 micrograms/ml proteins of fraction 1.0 mol/l and with 45 ng/ml proteins of fraction 3.0 mol/l, this last representing the most purified plasma fraction (about 8890-fold purification). The proteolytic activity of both plasma and purified plasma fractions was tested on Tos-Arg-OMe substrate which was hydrolyzed to a much higher degree by the most purified plasma fraction. Like the (Na-K)ATPase stimulation, the esterolytic activity was inhibited by protease inhibitors, the most effective to this regard being antithrombin.
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PMID:Identification and partial purification of a (Na-K)ATPase stimulating serine protease from plasma of insulin-dependent diabetics. 283 59

The egg white of marine turtle (Caretta caretta Linn.) and one species of tortoise (Geomyda trijuga trijuga Schariggar) contain a low molecular weight basic protein. It has been purified to homogeneity from the egg white of marine turtle and characterized in terms of its major physicochemical and chemical properties. The molecular weight of this protein calculated from gel filtration, sodium dodecyl sulfate-gel electrophoresis in the presence of urea, sedimentation-diffusion data, and amino acid composition is 4300. Its isoelectric point is at pH 11.1 and intrinsic viscosity is 0.038 dl g-1 in 0.2 M NaCl. It has a Stokes radius of 12.6 A and a diffusion coefficient of 16.50 x 10(-7) cm2 s-1. Analysis of the far-ultraviolet circular dichroic spectrum has shown that the basic protein contains 27% beta-pleated sheet and little or no alpha-helix. It possesses a single polypeptide chain of 40 amino acid residues with three disulfide bonds. It lacks serine, methionine, phenylalanine and carbohydrate moiety. It binds to DNA and stimulates ATPase activity due to its strong basicity. The complex of DNA-basis protein is partially resistant to the action of DNase.
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PMID:Purification and characterization of a low molecular weight basic protein from marine turtle egg white. 283 72

Two unique low molecular weight (531) compounds with both digoxin-like immunoreactivity and Na, K-ATPase inhibitory properties have been isolated from human plasma. One of these, digoxin-like substance 2, (DLIS-2), was studied by fast atom bombardment mass spectrometry and collisionally activated dissociation mass spectrometry/mass spectrometry. The fragment patterns were interpreted as being derived from a lysophosphatidyl serine containing a novel 19:4 fatty acid side chain. The molecular formula C25H42O9NP is consistent with these observations.
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PMID:Mass spectrometry studies of a novel digoxin-like substance (DLIS-2) isolated from human plasma ultrafiltrate. 283 88

The enzyme complex F1-ATPase has been isolated from bovine heart mitochondria by gel filtration of the enzyme released by chloroform from sub-mitochondrial particles. The five individual subunits alpha, beta, gamma, delta and epsilon that comprise the complex have been purified from it, and their amino acid sequences determined almost entirely by direct protein sequence analysis. A single overlap in the gamma-subunit was obtained by DNA sequence analysis of a complementary DNA clone isolated from a bovine cDNA library using a mixture of 32 oligonucleotides as the hybridization probe. The alpha, beta, gamma, delta and epsilon subunits contain 509, 480, 272, 146 and 50 amino acids, respectively. Two half cystine residues are present in the alpha-subunit and one in each of the gamma- and epsilon-chains; they are absent from the beta- and delta-subunits. The stoichiometry of subunits in the complex is estimated to be alpha 3 beta 3 gamma 1 delta 1 epsilon 1 and the molecular weight of the complex is 371,135. Mild trypsinolysis of the F1-ATPase complex, which has little effect on the hydrolytic activity of the enzyme, releases peptides from the N-terminal regions of the alpha- and beta-chains only; the C-terminal regions are unaffected. Sequence analysis of the released peptides demonstrates that the N terminals of the alpha- and beta-chains are ragged. In 65% of alpha-chains, the terminus is pyrrolidone carboxylic acid; in the remainder this residue is absent and the chains commence at residue 2, i.e. lysine. In the beta-subunit a minority of chains (16%) have N-terminal glutamine, or its deamidation product, glutamic acid (6%), or the cyclized derivative, pyrrolidone carboxylic acid (5%). A further 28% commence at residue 2, alanine, and 45% at residue 3, serine. The delta-chains also are heterogeneous; in 50% of chains the N-terminal alanine residue is absent. The sequences of the alpha- and beta-chains show that they are weakly homologous, as they are in bacterial F1-ATPases. The sequence of the bovine delta-subunit of F1-ATPase shows that it is the counterpart of the bacterial epsilon-subunit. The bovine epsilon-subunit is not related to any known bacterial or chloroplast H+-ATPase subunit, nor to any other known sequence. The counterpart of the bacterial delta-subunit is bovine oligomycin sensitivity conferral protein, which helps to bind F1 to the inner mitochondrial membrane.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Primary structure and subunit stoichiometry of F1-ATPase from bovine mitochondria. 286 55

Mutations in the uncB gene which encodes the a subunit of F1F0-ATPase in Escherichia coli were isolated and characterized. Eight mutations caused premature polypeptide chain termination. Two mutations were single amino acid substitutions resulting in the replacements of serine 206 with leucine (ser-206----leu) and histidine 245 with tyrosine (his-245----tyr). The ser-206----leu mutation does not alter F1 binding and allows ATP driven membrane energization at a low level. Stripping of F1 from membranes containing the ser-206----leu mutation does not render the membranes permeable to protons indicating impaired proton conductivity. The his-245----tyr mutation also blocks Fo-mediated proton conduction but has normal F1 binding properties. F1 bound to membranes with both ser-206----leu and his-245----tyr mutant a subunits is sensitive to dicyclohexylcarbodiimide. Apparently, both missense mutations impair proton conduction without altering assembly of the F1F0-ATPase complex. The direct involvement of the a subunit in proton translocation is discussed.
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PMID:Impaired proton conductivity resulting from mutations in the a subunit of F1F0 ATPase in Escherichia coli. 287 37

Peptides generated from enzymatic hydrolysis of chicken enolase and the alpha- and beta-subunits of bovine F1-ATPase were analyzed by mass spectrometry to determine the nature of their modified N-termini. In the case of chicken enolase, a peptide was isolated from a Staphylococcus aureus proteinase digest by HPLC and analyzed directly by fast atom bombardment mass spectrometry (FABMS). In conjunction with mass spectral evidence obtained from the methyl ester derivative and a secondary tryptic peptide, a structure is proposed containing an N-acetyl serine at the N-terminus. The alpha-subunit of bovine mitochondrial ATPase was chromatographed by HPLC after S. aureus proteinase digestion and a single peak was analyzed on the basis of predicted retention times. A Mr 716 was determined by FABMS and pyrrolidone carboxylic acid was deduced on the basis of its amino acid composition and partial Edman sequence data. The beta-subunit of ATPase produced a series of closely eluting peaks on HPLC after limited digestion with trypsin of the alpha 2 beta 2 complex. These peptides were analyzed by both Edman degradation and FABMS. These data showed the N-terminus to be frayed with N-terminal sequences beginning in pyro-Glu-Ala-Ser, Gln-Ala-Ser, Glu-Ala-Ser, Ala-Ser, and Ser but with no N-acetyl-Ser as was previously thought.
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PMID:Structural elucidation of N-terminal post-translational modifications by mass spectrometry: application to chicken enolase and the alpha- and beta-subunits of bovine mitochondrial F1-ATPase. 289 18

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


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