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:3.6.1.25 (triphosphatase)
1,529 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The Lubrol-dispersed guanylate cyclase from sea urchin sperm was purified and isolated essentially free of detergent by GTP affinity chromatography, DEAE-Sephadex chromatography, and gel filtration. After removal of the detergent, the enzyme remained in solution in the presence of 20% glycerol. The specific activity of the purified enzyme was about 12 mumol of guanosine 3':5'-monophosphate (cyclic GMP) formed - min-1 - mg of protein-1 at 30 degrees, an activity about 4600 times that of a soluble guanylate cyclase purified recently from Escherichia coli (Macchia V., Varrone, S., Weissbach, H., Miller, D.L., and Pastan, I. (1975) J. Biol. Chem. 250, 6214-6217). The cyclic GMP phosphodiesterase activity was negligible and adenosine 3':5'-monophosphate (cyclic AMP) phosphodiesterase was not detectable in the purified preparation. Cyclic AMP formation from ATP occurred at a rate of 0.002% of that of guanylate cyclase. In the absence of phosphodiesterase or guanosine triphosphatase inhibitors, 100% of the added GTP was converted to cyclic GMP. The purified enzyme required Mn2+ for maximum activity, the relative rates in the presence of Mg2+ or Ca2+ being less than 0.6% of the rates with Mn2+. The purified enzyme displayed classical Michaelis-Menten kinetics with respect to MnGTP (apparent Km is approximately equal to 170 muM) in contrast to the positively cooperative kinetic behavior displayed by the unpurified, detergent-dispersed, or particulate guanylate cyclase. The molecular weight of the purified enzyme was approximately 182,000 as estimated on Bio-Gel A-0.5m columns equilibrated in the presence or absence of 0.1 M NaCl. The unpurified, detergent-dispersed enzyme also migrated with an apparent molecular weight of 182,000 on columns equilibrated with 0.5% Lubrol WX and 0.1 M NaCl, but it migrated as a large aggregate (molecular weight is greater than 5 X 10(5)) on columns equilibrated in the absence of either the detergent of NaCl. After gel filtration, the unpurified, dispersed enzyme still yielded positive cooperative kinetic patterns as a function of MnGTP. Na dodecyl-SO4 gel electrophoresis of the enzyme after the DEAE-Sephadex or the gel filtration steps resulted in two major protein bands with estimated molecular weights of 118,000 and 75,000. Whether or not these protein bands represent the subunit molecular weights of guanylate cyclase is unknown at present.
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PMID:Sea urchin sperm guanylate cyclase. Purification and loss of cooperativity. 0 69

The dynamic properties of cross-bridge movement were investigated in glycerol-treated muscle fibers under various conditions by analyzing tension responses to two types of length change. First, the fiber bundles were stretched linearly with time for 0.3 s from the rest length (L0) by 2.5% of L0, suddenly released, then fixed at L0 (sudden release of the slow stretch). Second, they were stretched for 0.01 s by 2.5% of L0, then held at the plateau length (a quick stretch). 1. The transient tension responses following both length changes were divided into three phases: (i) very quick recovery of tension (0 approximately 0.05 s), (ii) quick recovery (0.05 approximately 0.3-0.4 s), and (iii) gradual recovery (0.3-0.4 s approximately several seconds). 2. The effects of activating conditions on the rates of the quick phases (0 approximately 0.3-0.4 s) were not associated with those on the nucleoside triphosphatase [EC 3.6.1.3] rates: the rates of the quick phases increased with increase in temperature and Mg2+-ATP concentration, with decrease in Ca2+ concentration, and also on replacement of Mg2+-ATP by Mg2+-ITP or Mn2+-ATP. Only a small amount of ADP, 0.07 mol per mol of myosin (Fig. 24 in the preceding paper), was liberated during the quick recovery phases. 3. The remaining slow tension recovery was concluded to be associated with one cycle of ATP splitting, and progressed very smoothly. This suggests that most of the cross-bridges do not exist in a synchronously dissociated state during one cycle of ATP splitting.
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PMID:Factors affecting the transient tension change after applying stepwise length change to glycerol-treated muscle fibers. Effects of temperature, divalent cations, and modification with p-chloromercuribenzoate. 47 41

RNA triphosphatase, RNA guanylyltransferase, and RNA (guanine-7)-methyltransferase activities are associated with the vaccinia virus mRNA capping enzyme, a heterodimeric protein containing polypeptides of Mr 95,000 and Mr 31,000. The genes encoding the large and small subunits (corresponding to the D1 and the D12 ORFs, respectively, of the viral genome) were coexpressed in Escherichia coli BL21 (DE3) under the control of a bacteriophage T7 promoter. Guanylyltransferase activity (assayed as the formation of a covalent enzyme-guanylate complex) was detected in soluble lysates of these bacteria. A 1000-fold purification of the guanylyltransferase was achieved by ammonium sulfate precipitation and chromatography using phosphocellulose and SP5PW columns. Partially purified guanylytransferase synthesized GpppA caps when provided with 5'-triphosphate-terminated poly(A) as a cap acceptor. In the presence of AdoMet the enzyme catalyzed concomitant cap methylation with 99% efficiency. Inclusion of S-adenosyl methionine increased both the rate and extent of RNA capping, permitting quantitative modification of RNA 5' ends. Guanylyltransferase sedimented as a single component of 6.5 S during further purification in a glycerol gradient; this S value is identical with that of the heterodimeric capping enzyme from vaccinia virions. Electrophoretic analysis showed a major polypeptide of Mr 95,000 cosedimenting with the guanylyltransferase. RNA triphosphatase activity cosedimented exactly with guanylyltransferase. Methyltransferase activity was associated with guanylyltransferase and was also present in less rapidly sedimenting fractions. The methyltransferase activity profile correlated with the presence of a Mr 31,000 polypeptide. These results indicate that the D1 and D12 gene products are together sufficient to catalyze all three enzymatic steps in cap synthesis. A model for the domain structure of this enzyme is proposed.
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PMID:Catalytic activity of vaccinia mRNA capping enzyme subunits coexpressed in Escherichia coli. 216 22

RNA triphosphatase, RNA guanylyltransferase, RNA (guanine-7)-methyltransferase, and transcription termination factor activities are associated with the mRNA capping enzyme from vaccinia virus. Purified vaccinia capping enzyme is a 6.5 S protein containing two subunits of Mr = 95,000 and Mr = 31,000. Although the RNA guanylyltransferase domain has been localized to the large subunit by virtue of the formation of a Mr = 95,000 covalent protein-GMP intermediate, the location of other functional domains within the protein and the catalytic role of individual subunits remain unclear. In the present study, limited proteolysis with trypsin was shown to convert the vaccinia capping enzyme into a form capable of generating a Mr = 59,000 enzyme-GMP complex. Purification of the trypsinized enzyme by glycerol gradient sedimentation resulted in the isolation of a 4.2 S fragment of the large subunit that retains RNA triphosphatase and RNA guanylyltransferase activities. This derivative, containing little or no small subunit (or fragments thereof), has lost the ability to catalyze methyl group transfer and to mediate transcription termination in vitro. Residual methyltransferase activity was found associated with a minor 5.2 S tryptic product that cosediments with a Mr = 21,000 fragment of the small enzyme subunit. A model for the organization of functional domains within the capping enzyme is suggested.
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PMID:Functional domains of vaccinia virus mRNA capping enzyme. Analysis by limited tryptic digestion. 254 18

Adenosine triphosphatase activated by divalent cations is apparently a component of the motile apparatus in flagella of Arbacia sperm, as judged by the activity of this enzyme in intact flagella, glycerol-extracted flagella, and soluble extracts prepared from flagella. However, the variation in the physical properties and in the amount of enzyme obtained after a variety of treatments suggests that additional components are involved in the motile mechanism. These features distinguish the soluble flagellar enzyme from adenosine triphosphatases of other motile cells.
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PMID:Flagellar adenosine triphosphatase from sea urchin sperm: properties and relation to motility. 417 99

1. The purification of an adenosine triphosphatase present in aqueous extracts of acetone-dried ox-heart mitochondria is described. 2. No evidence was found for the presence of more than one protein having adenosine-triphosphatase activity in these extracts. 3. The enzyme is less stable at 0 degrees than at 25 degrees but is stabilized by glycerol. 4. The activity is dependent on the presence of Mg(2+) or certain other bivalent metal cations. 5. The adenosine-triphosphatase activity of the Mg(2+)-activated enzyme is enhanced by 2,4-dinitrophenol. 6. The kinetics of Mg(2+) activation indicate that the ATP-Mg(2+) complex is the important substrate: free ATP and Mg(2+) are inhibitory. 7. This preparation of mitochondrial adenosine triphosphatase has many properties in common with the adenosine triphosphatase coupling factor from mitochondria (Racker, 1961).
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PMID:Preparation and general properties of a soluble adenosine triphosphatase from mitochondria. 422 55

Studies of the relationship between yield coefficient and growth rate, as affected by temperature of growth, in Escherichia coli have shown that, over a wide range of temperature, yield is relatively constant until the specific growth rate falls below about 0.2 hr(-1), at which point the yield begins to fall off precipitously. No intermediates of glucose metabolism in a form utilizable at higher temperatures could be found in the medium, and no toxic product was produced which limited growth. At 10 C, 37% of the carbon from glucose-UL-(14)C was assimilated into cellular material, whereas, at 30 C, 53% was assimilated. Cells grown at 10 C contained more carbohydrate than did cells grown at 37 C, and the glycogen-to-protein ratio of cells grown at 10 C was approximately three times higher than that of cells grown at 37 C. Adenosine triphosphatase activities of cells grown at 10 and 35 C were similar. Growth rates on glucose, glycerol, and succinate were quite similar at 10 C, but at 35 C growth was most rapid on glucose and slowest on succinate. The data suggest that the decrease in yield with decrease in temperature is a result of uncoupling of energy production from energy utilization.
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PMID:Effect of decreasing growth temperature on cell yield of Escherichia coli. 423 77

An RNA 5'-triphosphatase activity hydrolyzing gamma-phosphate from pppN-RNA was found to be associated with mRNA guanylyltransferase partially purified from rat liver nuclei. The activity specifically removed 32P as inorganic phosphate from [gamma-32P]pppA(pA)n, but not from [beta-32P]pppA(pA)n or from [gamma-32P]ATP. Free SH group(s) were required for its activity, and the reaction was inhibited by N-ethylmaleimide. Divalent cations were not required, but were rather inhibitory for the reaction. The RNA 5'-triphosphatase activity could not be separated from the guanylyltransferase activity through successive chromatographies on Sephadex G-150, CM-Sephadex and blue dextran-Sepharose columns. Both activities remained physically associated during sedimentation in glycerol density gradients after high salt treatment. The heat stability of the RNA 5'-triphosphatase activity was almost identical with that of the guanylyltransferase activity. These results indicate that the 69000 mol. wt. protein purified from rat liver nuclei as guanylyltransferase possesses both mRNA capping and RNA 5'-triphosphatase activities.
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PMID:Association of an RNA 5'-triphosphatase activity with RNA guanylyltransferase partially purified from rat liver nuclei. 613 53

Purified RNA guanylyltransferase . RNA (guanine-7-)methyltransferase complex from vaccinia virus contains RNA triphosphatase activity. The latter activity, which removes the gamma-phosphate from triphosphate-ended polyribonucleotides, co-chromatographed with the capping and methylating enzyme complex on seven different ion exchange or affinity columns and co-sedimented with the complex on a glycerol gradient. On a molar basis, the RNA triphosphatase was about 100 times more active than the associated RNA guanylyl-transferase. When the purified enzyme complex was incubated with poly(A) containing a 5'-triphosphate, removal of the gamma-phosphate preceded capping. Furthermore, there was no significant difference in the rate or extent of capping 5'-diphosphate- or 5'-triphosphate-ended poly(A). Physical association of the three enzymatic activities appears to be an efficient mechanism for carrying out the following successive steps in cap formation: (formula: see text).
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PMID:Modification of the 5' end of mRNA. Association of RNA triphosphatase with the RNA guanylyltransferase-RNA (guanine-7-)methyltransferase complex from vaccinia virus. 624 1

P protein of phage lambda and dnaB protein of Escherichia coli were isolated from (a) bacteria containing an inducible lambda P gene on a plasmid, and (b) phage-lambda-infected bacteria. P protein from both sources copurifies with part of the dnaB protein during four purification steps. A highly purified preparation contains the multimeric dnaB and the P protein in a complex as revealed by glycerol gradient centrifugation. The complex is composed of two major polypeptides. Their molecular weights of 52 000 and 26 000 are identical to those previously determined for the dnaB and P polypeptides, respectively. The complex contains a DNA-dependent ribonucleoside triphosphatase activity which can be inactivated by anti-dnaB globulin. Both the dnaB complementing and the ribonucleoside triphosphatase activities are partially masked by the P protein as shown by their stimulation following a treatment with sodium chloride and N-ethylmaleimide.
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PMID:Isolation of a complex between the P protein of phage lambda and the dnaB protein of Escherichia coli. 624 74


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