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:3.1.4.1 (phosphodiesterase)
18,767 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A procedure for the purification of phosphodiesterase from Crotalus venom on DEAE-cellulose at alkaline pH is described. The enzyme gives a single band in polyacrylamide gels and is free of contaminating nucleolytic enzymes. The molecular weight is about 115000. Concentration in an Amicon ultrafiltrator gave a highly concentrated active enzyme. Phosphodiesterase is relatively stable and can be stored at 4 degrees C in the presence of Mg2 and serum albumin for years. For the detection of contaminating endonuclease, an assay was used in which tRNA was the substrate and possible internal breaks were detected in polyacrylamide gel after denaturation. With bis(p-nitrophenyl) phosphate as substrate, 15mM Mg2 was necessary for optimal activity. The reaction remained linear for at least 15 min at 22 degrees C. At 45 degrees C, the liberation of p-nitrophenol was highest within 25 min of incubation. At 75 degrees C, inactivation of the enzyme occurred after 4 min.
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PMID:Purification and characterization of phosphodiesterase from Crotalus venom. 0 Feb 95

Digestion of tRNA by electrophoretically pure phosphodiesterase is limited to a short sequence of nucleotides at the 3'-terminus. On the average, four percent of all nucleotides can be released from tRNA. The optimum Mg2 concentration is 10mM and the optimum pH 9.2. The mode of action is a random attack by the enzyme on the substrate. The terminal AMP is completely removed at 15 degrees C after short incubation; about 400 mol of AMP were removed per min by 1 mol of enzyme. The following CMP residues are released much more slowly; at 15 degrees C incompletely, and at 37 degrees C more or less completely in 1 h. In about 50% of the tRNA molecules, the fourth nucleotide could be removed in very long incubations or with very high enzyme concentrations.
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PMID:Limited hydrolysis of tRNA by phosphodiesterase. 0 Feb 96

A procedure for the quantitative measurement of the O2'-methylnucleoside constitutents of RNA has recently been developed in this laboratory (Gray, M.W. Can. J. Biochem. 53, 735-746 (1975)). This assay method is based on the resistance of O2'-methylnucleoside 5'-phosphates (pNm) (generated by phosphodiesterase hydrolysis of RNA) to subsequent dephosphorylation by venom 5'-nucleotidase (EC 3.1.3.5). In the present investigation, two base-modified 5'-nucleotides, each displaying an unusual resistance to 5'-nucleotidase, have been identified. These compounds have been characterized by a variety of techniques as N2, N2-dimethylguanosine 5'-phosphate (pm2/2G) and 3-(3-amino-3-carboxypropyl)uridine 5'-phosphate (p4abu3U). Because of their resistance to 5'-nucleotidase, pm2/2G and p4abu3U are isolated along with the pNm in the mononucleotide fraction of venom hydrolysates of transfer RNA. Under hydrolysis conditions, the stability of p4abu3U is comparable to that of a pNm, allowing quantitative assay of the nucleotide. The proportion (mean +/- SD) of p4abu3U in venom hydrolysates of wheat embryo and Escherichia coli tRNA has been determined to be 0.35 +/- 0.03 (n=5) and 0.14 +/- 0.02 (n=4) mol%, respectively. The absence of p4abu3U in venom hydrolysates of yeast tRNA implies the absence of the corresponding nucleoside in yeast tRNA, in agreement with existing data. The variable recovery of pm2/2G from venom hydrolysates of wheat embryo and yeast tRNA indicates that under hydrolysis conditions, this base-modified nucleotide is only partially resistent to 5'-nucleotidase. The complete absence of pm2/2G in venom hydrolysates of E. coli tRNA is consistent with the known absence of N2, N2-dimethylguanosine in this RNA. These observations demonstrate that resistance to 5'-nucleotidase is a necessary but not sufficient criterion for concluding that a 5'-nucleotide is O2'-methylated. When applied to wheat embryo ribosomal RNA, the analytical methods described in this report failed to reveal any compound having the distinctive charge properties of p4abu3U. It therefore appears that 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine, recently characterized as a constituent of the 18 S rRNA of Chinese hamster cells (Saponara, A.G. & Enger, M.D. Biochim. Biophys. Acta 349, 61-77 (1974)), may not be present in wheat embryo ribosomal RNA.
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PMID:Modified 5'-nucleotides resistant to 5'-nucleotidase: isolation of 3-(3-amino-3-carboxypropyl) uridine 5'-phosphate and N2, N2-dimethylguanosine 5'-phosphate from snake venom hydrolysates of transfer RNA. 0 33

An acid ribonuclease has been purified from HeLa cell lysosomes. The specific activity of the RNase in lysosomes is 8-fold higher than that in nuclei and 15-fold higher than that in the postlysosomal fraction. The purified enzyme showed no detectable DNase, phosphodiesterase, phosphatase, or alkaline RNase activity. The acid RNase binds to Con A-agarose and is inferred to be a glycoprotein. It has a low isoelectric point at pH 3.0 to 3.5, and the optimal pH for activity is between 5.0 and 5.5. The enzyme requires no divalent cation for optimal activity and is totally inhibited by 1 mM Cu2+ or Hg2+. Monovalent cations including Na+, K+, and NH4+ stimulate the activity in low ionic strength buffer. The enzyme degrades rRNA faster than tRNA, and tRNA faster than poly(U); poly(A) and poly(C) are highly resistant. The products from rRNA are mostly oligonucleotides with 3'-phosphate ends. An acid RNase is also present in the lysosomes of L-cells grown in a medium free of serum; it is probably identical to the one described here.
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PMID:Acid ribonuclease from HeLa cell lysosomes. 3 88

4-Nitrophenyl and 2-napthyl monoesters of phenylphosphonic acid have been synthesized, and an enzyme catalyzing their hydrolysis was resolved from alkaline phosphatase of a commerical calf intestinal alkaline phosphatase preparation by extensive ion-exchange chromatography, chromatography on L-phenylalanyl-Sepharose with a decreasing gradient of (NH4) 2SO4, and gel filtration. Detergent-solubilized enzyme from fresh bovine intestine was purified after (NH4)2SO4 fractionation by the same technique. The purified enzyme is homogeneous by polyacrylamide gel electrophoresis and sedimentation equilibrium centrifugation. It has a molecular weight of 108,000, contains approximately 21% carbohydrate, and has an amino acid composition considerably different from that reported from alkaline phosphatase from the same tissue. The homogeneous intestinal enzyme, an efficient catalyst of phosphonate ester hydoolysis but not of phosphate monoester hydrolysis, was identified as a 5'-nucleotide phosphodiesterase by its ability to hydrolyze 4-nitrophenyl esters of 5'-TMP but not of 3'-TMP. Also consistent with this identification was the ability of the enzyme to hydrolyze 5'-ATP to 5'-AMP and PPi, NAD+ to 5'-AMP and NMN, TpT to 5'-TMP and thymidine, pApApApA to 5'-AMP, and only the single-stranded portion of tRNA from the 3'-OH end. Snake venom 5'-nucleotide phosphodiesterase also hydrolyzes phosphonate esters, but 3'-nucleotide phosphodiesterase of spleen and cyclic 3',5'-AMP phosphodiesterase do not. Thus, types of phosphodiesterases can be conveniently distinguished by their ability to hydrolyze phosphonate esters. As substrates for 5'-nucleotide phosphodiesterases, phosphonate esters are preferable to the more conventional esters of nucleotides and bis(4-nitrophenyl) phosphate because of their superior stability and ease of synthesis. Furthermore, the rate of hydrolysis of phosphonate esters under saturating conditions is greater than that of the conventional substrates. At substrate concentrations of 1 mM the rates of hydrolysis of phosphonate esters and of nucleotide esters are comparable and both superior to that of bis(4-nitrophenyl) phosphate.
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PMID:Hydrolysis of phosphonate esters catalyzed by 5'-nucleotide phosphodiesterase. 17 Sep 64

A phosphodiesterase characterized by a generally higher activity on 2'-5' than on 3'-5' phosphodiester bonds was isolated from mouse L cells treated with interferon. A similar enzyme was purified from mouse reticulocytes. The phosphodiesterase 2'-PDi splits the 2'-phosphate bond of pppA2'p5'A2'p5'A, the oligonucleotide activator of ribonuclease F. The level of phosphodiesterase 2'-PDi is increased by interferon treatment of L cells. The phosphodiesterase was also shown to degrade the C-C-A terminus of tRNA and to reduce the amino acid acceptance of tRNA in cell-free extracts, thereby causing a tRNA-reversible inhibition of mRNA translation.
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PMID:An interferon-induced phosphodiesterase degrading (2'-5') oligoisoadenylate and the C-C-A terminus of tRNA. 22 64

A ribonuclease (ribonucleate 3-pyrimidine-oligonucleotidohydrolase, EC 3.1.4.22) was purified 8300-fold from soluble fraction of beef brain and its properties were investigated. The enzyme is an endonuclease capable of hydrolyzing tRNA, rRNA, poly(C), but shows no activity towards poly(U), poly(A), and poly(G). The preparation is free of deoxyribonuclease, non-specific phosphodiesterase and phosphomonoesterase activity. The enzyme has a pH optimum of 7.6, is not heat stable, has a molecular weight of 25 000, and has a K-m of 134 mu rRNA and K-m of 1600 mug poly(C) per ml.
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PMID:Purification of an alkaline ribonuclease from soluble fraction of beef brain. 23 61

Splicing of tRNA precursors in extracts of Saccharomyces cerevisiae requires the action of two enzymes: a site specific endonuclease and a tRNA ligase. The tRNA ligase contains three distinct enzymatic activities: a polynucleotide kinase, a cyclic phosphodiesterase, and an RNA ligase. The polypeptide also has a high affinity pre-tRNA binding site based on its ability to form stable complexes with pre-tRNA substrates. To investigate the organization of functional enzymatic and binding elements within the polypeptide a series of defined tRNA ligase gene deletions were constructed and corresponding proteins were expressed in Escherichia coli as fusions with bacterial dihydrofolate reductase (DHFR). The DHFR/ligase derivative proteins were then efficiently purified by affinity chromatography. The complete ligase fusion protein retained enzymatic and binding activities which were unaffected by the presence of the DHFR segment. Examination of tRNA ligase deletion derivatives revealed that the amino-terminal region was required for adenylylation, while the carboxyl-terminal region was sufficient for cyclic phosphodiesterase activity. Deletions within the central region affected kinase activity. Pre-tRNA binding activity was not strictly correlated with a distinct enzymatic domain. A DHFR/ligase-derived protein lacking kinase activity efficiently joined tRNA halves. We postulate that this variant utilizes a novel RNA ligation mechanism.
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PMID:Deletion analysis of a multifunctional yeast tRNA ligase polypeptide. Identification of essential and dispensable functional domains. 185 Apr 8

Yeast tRNA ligase is one of two proteins required for the splicing of precursor tRNA molecules containing introns. The 95-kDa tRNA ligase has been purified to homogeneity from a strain of Escherichia coli which overexpresses the protein. The ligation reaction requires three enzymatic activities: phosphodiesterase, polynucleotide kinase, and ligase. By partial proteolytic digestion, we have produced fragments of tRNA ligase which contain the constituent activities. These results provide evidence for a model in which the three constituent activities of ligase are located in three distinct domains separated by protease-sensitive regions. We have also located the active adenylylated site in the ligase domains. It is lysine-114. The tRNA ligase sequence in this region has limited homology to the active-site region of T4 RNA ligase.
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PMID:Domain structure in yeast tRNA ligase. 220 62

Yeast tRNA ligase, from Saccharomyces cerevisiae, is one of the protein components that is involved in the splicing reaction of intron-containing yeast precursor tRNAs. It is an unusual protein because it has three distinct catalytic activities. It functions as a polynucleotide kinase, as a cyclic phosphodiesterase, and as an RNA ligase. We have studied the binding interactions between ligase and precursor tRNAs containing two photoreactive uridine analogues, 4-thiouridine and 5-bromouridine. When irradiated with long ultraviolet light, RNA containing these analogues can form specific covalent bonds with associated proteins. In this paper, we show that 4-thiouridine triphosphate and 5-bromouridine triphosphate were readily incorporated into a precursor tRNA(Phe) that was synthesized, in vitro, with bacteriophage T7 RNA polymerase. The analogue-containing precursor tRNAs were authentic substrates for the two splicing enzymes that were tested (endonuclease and ligase), and they formed specific covalent bonds with ligase when they were irradiated with long-wavelength ultraviolet light. We have determined the position of three major cross-links and one minor cross-link on precursor tRNA(Phe) that were located within the intron and near the 3' splice site. On the basis of these data, we present a model for the in vivo splicing reaction of yeast precursor tRNAs.
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PMID:Binding interactions between yeast tRNA ligase and a precursor transfer ribonucleic acid containing two photoreactive uridine analogues. 285 71


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