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)

A casein kinase activity, which copurifies with the H+-ATPase activity during isolation of plasma membranes Saccharomyces cerevisiae and during centrifugation of the solubilized membrane extract through a sucrose gradient, is separated from the Mr = 100,000 ATPase catalytic polypeptide by subsequent DEAE-cellulose chromatography. The purified casein kinase activity exhibits a low Km of 12 microM MgATP, is maximally stimulated by 6 mM free Mg2+, and is 50% inhibited by 300 microM Zn2+, by 7.5 micrograms of heparin/ml, and by 300 microM orthovanadate. It phosphorylates only seryl residues. The purified casein kinase contains two polypeptides of Mr = 45,000 and 39,000 which yield antibodies which do not cross-react to each other. The two polypeptides seem to originate from a precursor of Mr = 85,000 which is detected by both antibodies in partly purified fractions. In the absence of casein, a zinc and heparin-sensitive phosphorylation of the ATPase polypeptide is observed in partly purified ATPase fractions, and a peptide of similar mobility is phosphorylated, among others, in isolated plasma membranes. The purified ATPase activity is markedly inhibited by incubation in the presence of acid phosphatase. In agreement with a recent report that the purified active ATPase molecule is largely phosphorylated (Yanagita, Y., Abdel-Ghany, M., Raden, D., Nelson, N., and Racker, E. (1987) Proc. Natl. Acad. Sci. U. S. A. 894, 925-929) this data suggests that dephosphorylation leads to deactivation of ATPase activity.
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PMID:Characterization of a protein serine kinase from yeast plasma membrane. 289 78

The energy requirement for protein breakdown in Escherichia coli appears to be due to protease La, the lon gene product, which hydrolyzes proteins and ATP in a coupled process. This novel enzyme was investigated with small peptides, identified as substrates in the preceding manuscript. Although the degradation of proteins to acid-soluble material requires hydrolysis of a nucleoside triphosphate, cleavage of small fluorogenic substrates, such as glutaryl-Ala-Ala-Phe-methoxynaphthylamine, was found to require only binding of nucleotides to the enzyme. Nonhydrolyzable analogs of ATP, slowly hydrolyzed nucleotides, and even inorganic triphosphate and pyrophosphate stimulate the breakdown of these peptides but not of large proteins such as casein or serum albumin. In addition, vanadate, an inhibitor of the enzyme's ATPase activity, prevents protein degradation, but vanadate does not inhibit and can even stimulate peptide hydrolysis. Degradation of natural oligopeptides or of small polypeptides (less than 10,000 Da) also does not require hydrolysis of the nucleotide. Furthermore, although protein substrates promote ATP cleavage, the fluorogenic peptides inhibit this process. Also, no evidence was obtained for phosphorylation of the protease or of the substrate during ATP hydrolysis. These findings suggest that protein breakdown involves a cyclical series of reactions: 1) ATP binds to the protease and activates it allosterically, thus allowing peptide bond cleavage; 2) the hydrolysis of ATP must occur subsequently and should prevent further peptide bond cleavage until additional nucleoside triphosphates are bound; 3) with proteins as substrates, this reaction cycle probably occurs repeatedly until small peptides are generated.
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PMID:The role of ATP hydrolysis in the breakdown of proteins and peptides by protease La from Escherichia coli. 293 32

In addition to protease La (the lon gene product), Escherichia coli contains another ATP-dependent protease, Ti. This enzyme (approximately 340 kDa) is composed of two components, both of which are required for proteolysis. Both have been purified to homogeneity by conventional procedures using [3H]casein as the substrate. The ATP-stabilized component, A, has a subunit molecular weight of 80,000 upon gel electrophoresis in the presence of sodium dodecyl sulfate, but it behaves as a dimer (140 kDa) upon gel filtration. Component P, which is relatively heat stable, is inactivated by diisopropyl fluorophosphate and can be labeled with [3H] diisopropyl fluorophosphate. It has a subunit size of 23 kDa, but the isolated component behaves as a complex (260 kDa) of 10-12 subunits. The isoelectric point of component A is 7.0 and that of P is 8.2, and their amino acid compositions differ considerably. The purified enzyme has an ATPase activity that is stimulated 2-4-fold by casein and other protein substrates but not by nonhydrolyzed proteins. Component A also shows ATPase activity which can be stimulated by casein. Addition of component P (which lacks ATPase activity) inhibits basal ATP hydrolysis by A and makes this ATPase more responsive to casein. Although component P contains the serine active site for proteolysis, it shows no proteolytic activity in the absence of component A, Mg2+, and ATP or dATP. Other nucleoside triphosphates are not hydrolyzed and do not support proteolysis. Protease Ti has a Km for ATP of 210 microM for hydrolysis of both casein and ATP. Casein increases the Vmax for ATP without affecting the Km. A Mg2+ concentration of 5 mM is necessary for half-maximal rates of ATP and casein hydrolysis. Ca2+ and Mn2+ partially support these activities. Thus, protease Ti shares many unusual properties with protease La (e.g. coupled ATP and protein hydrolysis and protein-activated ATPase), but these functions in protease Ti are associated with distinct subunits that modify each other's activities.
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PMID:Protease Ti, a new ATP-dependent protease in Escherichia coli, contains protein-activated ATPase and proteolytic functions in distinct subunits. 296 16

A protein tyrosine kinase that phosphorylates both alpha and beta subunits of inactivated (Na+,K+)-ATPase from dog kidney was purified about 500-fold from Ehrlich ascites tumor cell membranes. The enzyme required divalent cations Mn2+, Mg2+, or Fe2+ but was inhibited by Cu2+ or Zn2+. The purified enzyme phosphorylated the beta subunit about five times faster than the alpha subunit of the (Na+,K+)-ATPase. The random polymer poly(Glu80Tyr20) was an excellent substrate while casein was only marginally phosphorylated. In contrast, the purified transforming gene product of Rous sarcoma virus phosphorylated all three substrates and the (Na+,K+)-ATPase was preferentially phosphorylated on the alpha subunit. The transforming gene product of Fujinami sarcoma visue and EGF receptor kinase from A431 cells phosphorylated (Na+,K+)-ATPase poorly whereas casein was an excellent substrate. The molecular weight of the partially purified protein tyrosine kinase from Ehrlich ascites tumor cells determined by gel filtration was about 60,000. One of two major phosphorylated phosphopeptides resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis had an Mr of 60 kDa, thus suggesting that it might be the autophosphorylated protein tyrosine kinase. A phosphatase that hydrolyzes phosphorylated histones or poly(Glu80Tyr20) was partially purified from the same membrane.
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PMID:A tyrosine-specific protein kinase from Ehrlich ascites tumor cells. 302 71

The ATP-binding component (Component II, hereafter referred to as ClpA) of a two-component, ATP-dependent protease from Escherichia coli has been purified to homogeneity. ClpA is a protein with subunit Mr 81,000. It has an intrinsic ATPase activity and activates degradation of protein substrates only in the presence of a second component (Component I, hereafter referred to as ClpP), Mg2+, and ATP. The amount of ClpA varies by less than a factor of 2 in cells grown in different media and at temperatures from 30 to 42 degrees C. ClpA does not appear to be a heat-shock protein since its synthesis is not dependent on htpR. Antibodies against purified ClpA were used to identify lambda transducing phage bearing the clpA gene. The cloned gene contains a DNA sequence expected to code for the first 28 amino acids of ClpA, which were determined by protein sequencing of purified ClpA. The clpA gene in the phage was mutated by insertion of delta kan defective transposons and the mutations were transferred to E. coli by homologous recombination. The clpA gene was mapped to 19 min on the E. coli chromosome. Mutant cells with insertions early in the gene produce no ClpA protein detectable in Western blots, and extracts of such mutant cells have no detectable ClpA activity. clpA- mutants grow well under all conditions tested and are not defective in turnover of proteins during nitrogen starvation nor in the turnover of such highly unstable proteins as the lambda proteins O, N, and cII, or the E. coli proteins SulA, RcsA, and glutamate dehydrogenase. The degradation of abnormal canavanine-containing proteins is defective in clpA mutants especially in cells that also have a lon- mutation. Extracts of clpA- lon- cells have ATP-dependent casein degrading activity.
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PMID:The two-component, ATP-dependent Clp protease of Escherichia coli. Purification, cloning, and mutational analysis of the ATP-binding component. 304 6

The energy requirement for protein breakdown in Escherichia coli has generally been attributed to the ATP-dependence of protease La, the lon gene product. We have partially purified another ATP-dependent protease from lon-cells that lack protease La (as shown by immunoblotting). This enzyme hydrolyzes [3H]methyl-casein to acid-soluble products in the presence of ATP and Mg2+. ATP hydrolysis appears necessary for proteolytic activity. Since this enzyme is inhibited by diisopropyl fluorophosphate, it appears to be a serine protease, but it also contains essential thiol residues. We propose to name this enzyme protease Ti. It differs from protease La in nucleotide specificity, inhibitor sensitivity, and subunit composition. On gel filtration, protease Ti has an apparent molecular weight of 370,000. It can be fractionated by phosphocellulose chromatography or by DEAE chromatography into two components with apparent molecular weights of 260,000 and 140,000. When separated, they do not show proteolytic activity. One of these components, by itself, has ATPase activity and is labile in the absence of ATP. The other contains the diisopropyl fluorophosphate-sensitive proteolytic site. These results and the similar findings of Katayama-Fujimura et al. [Katayama-Fujimura, Y., Gottesman, S. & Maurizi, M. R. (1987) J. Biol. Chem. 262, 4477-4485] indicate that E. coli contains two ATP-hydrolyzing proteases, which differ in many biochemical features and probably in their physiological roles.
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PMID:Escherichia coli contains a soluble ATP-dependent protease (Ti) distinct from protease La. 330 28

The purification and properties of a protein serine kinase (PK-P) extracted with Triton X-100 from membranes of bakers' yeast are described. The enzyme is virtually inactive unless either a histone or a heat-stable polypeptide from yeast membranes and Mg2+ are added. Other divalent cations substitute for Mg2+ poorly or not at all; most of them, including Mn2+, inhibit when added in the presence of 5 mM Mg2+. The enzyme is unstable but can be stabilized by addition of 0.1% Triton X-100 and 20% glycerol. The final preparation shows, on silver-stained electrophoresis gels, two major bands (Mr 41,000 and 35,000). According to gel filtration the molecular weight of the active protein is about 75,000. Of the two subunits, only the smaller one appears to be autophosphorylated. In addition to casein, the enzyme phosphorylates several proteins including the H+-ATPase (Mr 100,000) in the yeast plasma membrane. In order to demonstrate the phosphorylation of the ATPase (up to 0.9 equivalents), exposure of the latter to an acid phosphatase was required. Other phosphorylated proteins include mRNA cap-binding protein from mammalian erythrocytes and yeast, a glucocorticoid receptor protein, and a preparation of the guanine nucleotide-binding proteins Gi and Go from brain. A partial purification of a natural activator from yeast plasma membranes is described.
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PMID:Polypeptide-dependent protein kinase from bakers' yeast. 354 2

A new ATP-dependent, casein-degrading proteolytic complex has been identified and partially purified from Escherichia coli. The proteolytic complex can be isolated from wild-type cells as well as from mutants in which the gene for the ATP-dependent Lon protease is deleted. The complex consists of at least two components (components I and II) that can be separated from each other (and from wild-type Lon protease) by phosphocellulose chromatography. Neither component has casein-degrading activity when added separately to assay solutions with or without ATP. Both components must be present simultaneously for casein degradation to occur. Of the nucleotides tested, only ATP activates the proteolytic complex, and the ATP must be present continuously for degradation to occur. Component II copurifies with an ATPase activity and binds to a Type 4 ATP affinity column. ATP protects component II from heat inactivation, suggesting that component II interacts with ATP. Proteolysis was not inhibited by any serine protease inhibitors but was inhibited by reagents such as the organomercurial Neohydrin and N-ethylmaleimide, which react with sulfhydryl groups. Our data provide convincing evidence that E. coli possesses a previously undescribed proteolytic system composed of at least two complementary components and absolutely dependent on ATP.
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PMID:A multiple-component, ATP-dependent protease from Escherichia coli. 354 8

Mitochondria from bovine hearts were fractionated by three different procedures and the fractions were characterized by marker enzymes. Highly purified outer membranes, membrane vesicles, and inner membranes, as well as two high-speed soluble fractions, were obtained. Azide (or oligomycin) resistant ATPase was not found to be a marker for outer membranes. The data were consistent with the association of the protein kinase activity with the soluble matrix of the mitochondria. Activity was highest with histone H2B as the substrate, with histone H1 next in preference. In contrast to the mitochondrial protein kinases studied previously, protamine, casein, and phosvitin were very poor substrates and there was no detectable phosphorylation of pyruvate dehydrogenase. Activity was stimulated by cAMP but not by cGMP, calmodulin, or phosphatidylserine--diolein, with or without Ca2+. Two cAMP-dependent isozymes were separated from the soluble fraction of the mitochondria by chromatography on DE-52 columns. Phosphorylation of histone H2B by the isozymes was inhibited by 98% by Kemptide.
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PMID:cAMP-dependent protein kinase isozymes with preference for histone H2B as substrate in mitochondria of bovine heart. 382 13

We have purified an ATP-dependent protease with protein-dependent ATPase activity from bovine adrenal cortex mitochondria to near homogeneity. The subunit molecular weight is 108,000 and the enzyme appears to be a hexamer with approximately identical subunits. Based on the experiments using various nucleoside triphosphates and their related compounds, it is concluded that hydrolysis of the high-energy bond in nucleoside triphosphates is not an absolute requirement for proteolysis. Nucleotide specificity of this enzyme varies, depending on the protein or peptide substrates used. When casein was the substrate, ATP and dATP were quite effective, but other nucleotides were not. When insulin and angiotensinogen were used as substrate, ATP, other nucleoside triphosphates, ADP, inorganic triphosphate, pyrophosphate, and phosphate were effective. One of the cleaving linkages hydrolyzed by this enzyme was revealed to be the Leu-Leu bond of angiotensinogen. However, the specificity appears to be broad in view of the hydrolysis pattern of glucagon.
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PMID:Adrenal cortex mitochondrial enzyme with ATP-dependent protease and protein-dependent ATPase activities. Purification and properties. 390 33


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