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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 nucleoside triphosphate-stimulated efflux of RNA from isolated nuclei was studied under a range of conditions, and the effects of these conditions on the process were compared with the properties of the nucleoside triphosphatase located in the pore complex. A marked similarity between the rate of efflux and the rate of nucleoside triphosphate hydrolysis was apparent, in terms of substrate specificity, sensitivity to treatment with insolubilized trypsin, kinetics and the effects of increased ionic strength and of many inhibitors. These results are taken, in view of earlier evidence, to suggest that the activity of the nucleoside triphosphatase is a prerequisite for nucleo-cytoplasmic RNA transport in vivo. There are some indications that the nuclear-envelope lipid is also involved in regulating the efflux process.
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PMID:Importance of mammalian nuclear-envelope nucleoside triphosphatase in nucleo-cytoplasmic transport of ribonucleoproteins. 22 28

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

Limited tryptic digestion of Escherichia coli transcription termination factor rho [an RNA-dependent nucleoside triphosphatase (NTPase)] yields predominantly two fragments (f1 and f2) when the protein is bound to both poly(C) and ATP. The apparent molecular masses of the two fragments are 31 kDa for f1 and 15 kDa for f2, adding up to the molecular mass of the intact rho polypeptide chain (46 kDa). Sequence analysis of the amino termini demonstrates that f1 is derived from the amino-terminal portion of rho and that the trypsin cleavage that defines f2 occurs at lysine-283. These results suggest that, in the liganded (activated) form, the native rho protein monomer is organized into two distinct structural domains that are separable by a single proteolytic cleavage. The f1 fragment, purified from NaDodSO4/polyacrylamide gels and renatured, binds poly(C) but the f2 fragment does not; neither regains any ATPase activity. ATP- and polynucleotide-dependent changes in the rate of proteolysis and in the character of the fragments produced suggest that rho undergoes a series of conformational transitions as a consequence of RNA binding, NTP binding and NTP hydrolysis. The rate of loss of rho ATPase activity and of intact rho monomers is slower in the presence of adenosine 5'-[gamma-thio]triphosphate than in the presence of either ATP or ADP, indicating that the hydrolysis of ATP may result in different conformational effects than does the binding of this ligand. These findings are discussed within the context of recent models of rho-dependent transcription termination.
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PMID:Escherichia coli transcription termination factor rho has a two-domain structure in its activated form. 258 Mar 3

The major nucleoside triphosphatase (NTPase) of rat liver nuclear scaffold (NS) or envelope, which is thought to participate in nucleocytoplasmic transport, has been identified via photoaffinity labeling as a 46-kDa polypeptide. This 46-kDa protein was purified by SDS-polyacrylamide gel electrophoresis and cleaved with trypsin. The resulting peptides were purified by HPLC and five were microsequenced. All five peptides appear to be derived from the N-terminal region of lamins A/C. Subsequent experiments with photolabeled NS showed that the 46-kDa polypeptide was selectively immunoprecipitated by antiserum specific to lamins A/C and by affinity-purified anti-lamin antibodies. Photolabeling of nuclei prepared in the presence of protease inhibitors showed predominant labeling of the 46-kDa polypeptide, suggesting that it is an integral nuclear constituent and not an artifact produced during NS preparation. Use of protease inhibitors throughout purification of NS increased the specificity of photolabeling of the 46-kDa band by significantly reducing photolabeling of smaller molecular weight components, which arise by proteolysis. Anti-lamin antibodies also produced a significant inhibition of NTPase activity in NS. These results suggest that the N-terminal portion of lamins A/C represents the 46-kDa NTPase, which, according to previous reports, may participate in RNA transport.
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PMID:The 46-kDa nucleoside triphosphatase of rat liver nuclear scaffold represents the N-terminal portion of lamins A/C. 283 27

The step which requires the hydrolysis of a nucleoside triphosphate for translocation of a protein across microsome was investigated by studying translocation uncoupled from translation using two truncated products of invertase: one product contains the first 262 amino acids of the secreted invertase (Inv262); the other, the first 104 amino acids (Inv104). The truncated products were translated from RNA transcripts without a stop codon. It is demonstrated that the translated products contain an associated ribosome, and the associated ribosome is essential for the posttranslational translocation phenomenon. Also, it is demonstrated that binding of Inv262 to microsomal vesicles made from the cell wall-less mutant of Neurospora crassa does not require the hydrolysis, or the presence, of a nucleoside triphosphate. However, the posttranslational translocation across the membrane, as monitored by glycosylation of the translocated polypeptide chain, does. The data suggest that a nucleoside triphosphatase activity is associated with the translocation of invertase across the membrane. Furthermore, it is demonstrated that binding, and subsequent translocation across the membrane, is dependent on trypsin-sensitive membrane component(s).
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PMID:Translocation of a fragment of invertase across microsomal vesicles isolated from Neurospora crassa requires the hydrolysis of a nucleoside triphosphate. 297 55

1. The action of trypsin, chymotrypsin and subtilisin on the adenosine-triphosphatase and actin-combining activities, as measured by viscometric means, of H-meromyosin were compared. 2. Subfragment 1 produced by prolonged tryptic digestion has a molecular weight of 129000. 3. The preparations isolated by gel filtration and actin combination were shown to be similar. 4. Subfragment-1 preparations possess appreciably higher adenosine-triphosphatase activities than H-meromyosin when related to total nitrogen. 5. Chromatographic and gelfiltration studies indicated that adenosine-triphosphatase activity is not distributed uniformly in all fractions of subfragment 1. 6. The Ca(2+)-activated adenosine triphosphatase of subfragment 1 was stimulated by thiol reagents in a similar fashion to myosin and H-meromyosin. 7. Subfragment 1 differed from myosin and H-meromyosin in that its adenosine triphosphatase was only slightly activated by Mg(2+) in the presence of actin. 8. A subfragment-1-like component was obtained by chymotryptic digestion of H-meromyosin. 9. The results obtained from enzymic and hydrodynamic studies and from amino acid analyses are compatible with the concept of one molecule of H-meromyosin giving rise to one molecule of subfragment 1 on proteolytic digestion.
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PMID:The biological activity of subfragment 1 prepared from heavy meromyosin. 422 74

The partially purified preparation of messenger RNA guanylyltransferase from Artemia salina contains, as in the case of the rat liver enzyme (Yagi, Y., Mizumoto, K., and Kaziro, Y. (1983) EMBO J. 2, 611-615), the RNA 5'-triphosphatase activity which specifically removes the gamma-phosphoryl group from the 5'-triphosphoryl end of the newly synthesized mRNA molecule. The enzyme consists of a single polypeptide chain of Mr = 73,000 and forma a covalent enzyme-GMP complex as an intermediate for the guanylyltransferase reaction. Upon limited hydrolysis with trypsin, the enzyme-[32P]GMP complex is converted to a smaller 32P-containing fragment of Mr = 44,000. When the free enzyme, not complexed with GMP, is digested with trypsin under the same condition as above, the digests retain almost full activities of both guanylyltransferase and RNA 5'-triphosphatase and can form an enzyme-[32P]GMP complex of the size of Mr = 44,000 on incubation with [alpha-32P]GTP. Functional domains harboring the activities of guanylyltransferase and RNA 5'-triphosphatase are separated by gel filtration on a Sephacryl S-200 column at positions corresponding to Mr = 44,000 and 20,000, respectively. They can be separated completely from each other by CM-Sephadex column chromatography. While the native, undigested enzyme can transfer the GMP moiety to mRNA molecules with either triphosphoryl (pppN-) or diphosphoryl (ppN-)5'terminal, the purified Mr = 44,000 domain with the guanylyltransferase activity can utilize only the latter as an acceptor.
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PMID:Limited tryptic digestion of messenger RNA capping enzyme from Artemia salina. Isolation of domains for guanylyltransferase and RNA 5'-triphosphatase. 614 51

Upon incubation with trypsin, the adenosine-5'-triphosphatase (ATPase) activity of the nucleotide-depleted F1 is first rapidly and slightly activated and then slowly inactivated. The first phase is simultaneous with the conversion of the alpha subunit into an alpha' fragment which migrates between alpha and beta on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The second phase is related to the proteolysis of the three main subunits, alpha', beta, and gamma. Preincubation of the enzyme with low concentrations of adenosine 5'-diphosphate (ADP) or adenosine 5'-triphosphate (ATP) does not modify the slight increase of activity but efficiently prevents the inactivation induced by trypsin. The alpha leads to alpha' conversion is not affected whereas the further proteolysis of alpha', beta, and gamma does not occur. On the contrary, even high concentrations of GDP only slightly lower the trypsin-induced inactivation. The presence of endogenous tightly bound nucleotides also partially lowers the sensitivity to trypsin since F1 is less rapidly inactivated and proteolyzed than the nucleotide-depleted F1. Phosphate, at high concentrations, both slows down the first phase of activation and simultaneous alpha leads to alpha' conversion and prevents the second phase of inactivation and proteolysis of the main subunits. Pretreatment of the nucleotide-depleted F1 with trypsin under conditions where the ATPase activity is largely inhibited only slightly modifies, however, the hysteretic behavior of the enzyme: the ADP binding and the concomitant hysteretic inhibition of the residual activity are not markedly diminished. The purified ATPase-ATP synthase complex binds very few ADP's and is not hysteretically inhibited. Its ATPase activity is rapidly activated but not further inhibited by trypsin. Preincubation of the complex with ADP does not modify the effects of trypsin.
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PMID:Use of trypsin to monitor conformational changes of mitochondrial adenosinetriphosphatase induced by nucleotides and phosphate. 622 Jul 37

Adenosine triphosphatase activity of U. urealyticum is an integral membrane-bound protein which cannot be detached from the membrane by mild treatment with EDTA in low-ionic strength media nor by ionic detergents which rapidly inactivated the enzyme. The enzyme was Mg++ dependent; Mn++ and Co++ could replace Mg++ to some extent. A slight stimulatory effect was also exerted by sodium and lithium. The enzyme showed a nucleotide triphosphatase activity, but ADP was hydrolyzed at close to 40% the rate of ATP and other nucleotide monophosphatase were hydrolyzed at a very slow rate. Oubain and oligomycin did not inhibit the adenosine triphosphatase activity, whereas DCCD, NBD-Cl and several sulfhydryl-blocking reagents strongly reduced its activity. The enzyme could not be stimulated by trypsin pretreatment. It seems that the complex enzyme is tightly linked to the lipid bilayer of the membrane and differs in many aspects from the F0-F1 (Mg++, Ca++)-ATPase of bacteria.
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PMID:Adenosine triphosphatase activity of Ureaplasma urealyticum. 628 75

The Escherichia coli strain D2216 contains a kirromycin-resistant elongation factor Tu [EF-Tu(D2216); Fischer, E., Wolf, H., Hantke K., & Parmeggiani, A. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 4341-4345]. This stain grows much more slowly than wild-type E. coli strains and contains less than half the amount of EF-Tu. On isoelectric focusing, the whole cell lysate of strain D2216 as well as pure, crystalline EF-Tu(D2216) comprises only a single species indistinguishable from wild-type EF-Tu. In poly(uridylic acid)- [poly(U)] directed poly(phenylalanine) synthesis, enzymatic binding of aminoacyl transfer ribonucleic acid to the ribosome, and susceptibility to trypsin digestion, EF-Tu(D2216) behaves similarly to the EF-Tu from wild-type strains. Kirromycin, which increases the sensitivity to trypsinization of wild-type EF-Tu, has no effect on mutant EF-Tu. In poly(U)-directed poly(phenylalanine) synthesis, partially trypsinized EF-Tu(D2216) displays a 7-fold reduction of its kirromycin resistance as compared to the intact EF-Tu(D2216). This is approximately 300 times less sensitive to the antibiotic than wild-type EF-Tu. The EF-Tu(D2216), purified and crystallized, exhibits a guanosine 5'-triphosphatase activity in the absence of any other physiological effector or kirromycin. This activity is not a contaminant, since it can be selectively stimulated by ribosomes and is inactivated by temperature exactly in the same way as the guanosine 5'-diphosphate binding activity of Ef-Tu(D2216). We conclude that, as consequence of the mutation, the catalytic center of EF-Tu(D2216)-dependent guanosine 5'-triphosphate hydrolysis undergoes spontaneous activation.
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PMID:Characterization of a kirromycin-resistant elongation factor Tu from Escherichia coli. 701 93


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