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 method is described for the extensive purification of acid deoxyribonuclease (acid DNase) and its specific inhibitor from beef liver, the existence of which had been only supported by indirect evidence. By the use of insolubilized acid deoxyribonuclease, eight other proteins interacting with the enzyme have been detected. One of them (molecular weight, 59,000) was identified as responsible for phosphodiesterase activity which is often a contaminant of DNase preparations. Acid DNase (free of phosphodiesterase) and its inhibitor have been obtained as homogeneous proteins, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of acid DNase and its inhibitor are, respectively, 26,500 and 21,500; those of other proteins range from 17,000 to 112,000. The properties of beef liver acid DNase are similar to those described for the enzymes extracted from other sources. The same alteration of DNase kinetics by this inhibitor, as that previously demonstrated with an impure protein has been confirmed; the sigmoidal shape observed at pH 5 for the plot of initial rate versus substrate concentration progressively disappears with increasing pH. We have also demonstrated that RNA, which inhibits the acid DNase through a competitive binding to the catalytic site, is able, like the substrate, to reverse the binding of inhibitor to the enzyme.
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PMID:Protein inhibitor of acid deoxyribonucleases. Improved purification procedure and properties. 0 Mar 96

Human urine RNase was purified about 2000-fold. The preparation is free from phosphatase, phosphodiesterase and DNase activities. On electrophoresis through polyacrylamide gel at pH 8.3, it migrates toward the anode and stains with periodic acid-Schiff reagent, suggesting that it is acidic and glycoprotein in nature. Its isoelectric point is at pH 4.1. It has a molecular weight of about 21,500. It is thermostable at pH 4.2 and thermolabile at pH 8.5. It has a pH optimum at 6.5. It exhibits highest preference for cytidine 3'-phosphate linkages. Its activity on poly (C) is endonucleolytic. It cleaves poly (C) via intramolecular transphosphorylation. It has no action on cytidine 2': 3'-cyclic phosphate or uridine 2':3'-cyclic phosphate. Its rate of hydrolysis of poly (U) is less than 2% of that of poly C). Poly (A) and poly (G) are totally inert to its action. Its action on poly (C) is inhibited by poly (G), poly (A) and poly (U). It differs from bovine pancreatic Rnase A in its physical, chemical and catalytic properties. It is, however, similar to human serum and pancreatic RNase in all its properties, suggesting that pancreas is its likely source.
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PMID:Purification and properties of a ribonuclease in human urine that hydrolyses polycytidylic acid. 2 Jun 15

By sequential acid treatment, gel filtration and KM-cellulose sorption a 18--20-fold purified preparation of ribonuclease with a yield of 50--60% was obtained from the culture liquid filtrate of Actinomyces rimosus 994. The preparation had a high specific activity of 450,000--600,000 units/mg protein, contained 85--98% protein, insignificant amounts of carbohydrates and hydroxytetracycline, and no quantities of DNase, phosphomonoesterases, phosphodiesterase or proteases. In RNA degradation (preparation of the total yeast RNA of the Sigma Co.) optimal results were obtained at 50 degrees C and pH 7.0--7.2 in phosphate buffer and 7.6--8.0 IN Tris-HCl buffer. The preparation was stable at high temperatures (80--100 degrees) in the wide pH range and during storage in the lyophilized form and in buffer solutions. RNase effect was inhibited by zinc, copper, iron and cobalt cations and activated by beta-mercaptoethanol, citrate and EDTA. Protamine sulphate and urea in low concentrations (0.01% and 1--4 M, respectively) accelerated and in high concentrations (1% and 8 M, respectively) terminated the enzyme reaction. With respect to many properties RNase from Act. rimosus 994 was similar to extracellular RNases, produced by other actinomycetes and fungi.
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PMID:[Preparation of extracellular ribonuclease form Actinomyces rimosus 994]. 3 16

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

The paper describes a method for isolating alkaline ribonuclease from the culture liquid Bacillus subtilis KP 349 which involved: submerged cultivation of the producer on complex and synthetic nutrient media with optimized RNase activity, acid treatment of the total culture liquid, and filtration through perlite. Further treatment may include either spray drying of the culture liquid filtrate or its concentration in a vacuum evaporator, dialysis of the concentrate against distilled water, and dialyzate lyophilization. As a result, commercial RNase preparations with activities of 30--60 thous. and 160--300 thous. units/g, respectively, were obtained. The enzyme purification was carried out by chromatography and rechromatography on phosphocellulose columns. The resultant RNase of Bac. subtilis had a specific activity of 41--44 thous. units/mg protein, contained no nonspecific phosphodiesterase, DNase, acid or alkaline phosphomonoesterases.
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PMID:[Preparation of extracellular ribonuclease from Bacillus subtilis]. 10 17

The effect of several enzymes of the DNA metabolism of Escherichia coli on the biological activity of native and single-stranded T7 DNA was studied by transfection of lysozyme-EDTA spheroplasts prepared from various E. coli mutants. It is shown that the presence of the recBC DNase in the recipient cells decreases the infectivity of native and denatured DNA by about 100- and 10-fold, respectively. Lack of exonuclease I did not stimulate transfection by single-stranded DNA. Separated light (l) and heavy (r) strands of T7 DNA are fully infective, with a linear dependence on DNA concentrations, whereas heat-denatured DNA shows a two-hit kinetics. Single-stranded DNA was observed to depend on a functional DNA polymerase III for infectivity in polAB cells, whereas transfection with native T7 DNA was independent of the host DNA polymerases. The results are discussed with respect to the mode of T7 DNA replication.
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PMID:In vivo effects of recBC DNase, exonuclease I, and DNA polymerases of Escherichia coli on the infectivity of native and single-stranded DNA of bacteriophage T7. 32 5

Poly ADP-ribosylation of two mouse lymphoma cell lines, L5178Y (LS) and the radiation and alkylating agent resistant derivative AII, was investigated by uptake of [3H]NAD by permeabilised cells into acid-precipitable material that was sensitive to phosphodiesterase but insensitive to DNase and RNase. Basal activities in both lymphoma lines were 3-4-fold greater than in mouse L1210 leukaemia cells. However, total endogenous poly (ADP-R) polymerase activity in both L5178Y cell lines, stimulated by a large excess of DNase in the presence of Triton X-100, was less than half the activity in L1210 cells. Doses of N-methyl-N-nitrosourea (MNU) that produced 20-50% survival of colony-forming units increased poly (ADP-R) in both lymphoma lines by only 25% compared with 377% in L1210 cells when synthesis was measured immediately after a 30-min exposure of MNU. During the first 24 h after MNU AII cells produced a peak of activity that was not seen with LS cells. A second peak was seen in both cell lines between 24 and 48 h following MNU. Concentrations of 3-aminobenzamide (3AB) above 2.5 mM inhibited colony-forming ability of lymphoma cells and equally inhibited uptake of [14C]formate into protein, RNA and DNA indicating that 3AB behaves as a general metabolic poison. Concentrations of 3AB in the toxic range of 3-10 mM inhibited poly (ADP-R) synthesis but no degradation of the polymer was observed. Non-toxic concentrations of 3AB potentiated cell killing by MNU to a similar degree in both lymphoma cell lines. In conclusion, we have found little evidence to support the hypothesis that the differential sensitivity of LS and AII is related to poly ADP-ribosylation. Compared with other mouse cells, L5178Y cells appear deficient in poly (ADP-R) polymerase and poly (ADP-R) glycohydrolase activities.
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PMID:Poly (ADP-ribose) metabolism in alkylated mouse L5178Y cells. 299 Jul 53

The vast majority of nuclease activity in yeast mitochondria is due to a single polypeptide with an apparent molecular weight of 38,000. The enzyme is located in the mitochondrial inner membrane and requires non-ionic detergents for solubilization and activity. A combination of heparin-agarose and Cibacron blue-agarose chromatography was employed to purify the nuclease to approximately 90% homogeneity. The purified enzyme shows multiple activities: 1) RNase activity on single-stranded, but not double-stranded RNA, 2) endonuclease activity on single- and double-stranded DNA, and 3) a 5'-exonuclease activity on double-stranded DNA. Digestion products with DNA contain 5'-phosphorylated termini. Antibody raised against an analogous enzyme purified from Neurospora crassa (Chow, T. Y. K., and Fraser, M. (1983) J. Biol. Chem. 258, 12010-12018) inhibits and immunoprecipitates the yeast enzyme. This antibody inhibits 90-95% of all nuclease activity present in solubilized mitochondria, indicating that the purified nuclease accounts for the bulk of mitochondrial nucleolytic activity. Analysis of a mutant strain in which the gene for the nuclease has been disrupted supports this conclusion and shows that all detectable DNase activity and most nonspecific RNase activity in the mitochondria is due to this single enzyme.
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PMID:Purification and properties of the major nuclease from mitochondria of Saccharomyces cerevisiae. 328 39

Putrescine, spermidine, spermine and two unknowns designated as A and B were detected in first seedling leaves of barley (Hordeum vulgare L. var. Wolfe). The levels of these polyamines in first seedling leaves from 4-day-old barley plants grown in darkness or in light were comparable and did not change significantly after exposure of dark grown plants to light for 24 h. No significant consistent changes in the amounts of above polyamines, except perhaps decline in spermidine, were noted during senescence of intact or excised first seedling leaves of barley and this spermidine decline was suppressed during retardation of senescence of excised leaves by 10 mg/l kinetin in the dark. In addition, putrescine, spermidine, spermine, cadaverine and diaminopropane (0.2 mM, 1 mM, 10 mM) had no effect on senescence of excised barley leaves in the dark and both spermine and spermidine induced bleaching of the leaves in the light. Both spermine and spermidine (approx. 10 mM) inhibited RNase and DNase activities but stimulated phosphodiesterase activity (assayed with bis-p-nitrophenyl phosphate as substrate) in crude soluble extracts from barley leaves. Purified snake venom phosphodiesterase activity assayed with RNA as substrate was, however, stimulated by 300-400% by 7-14 mM spermine or spermidine indicating similar possibilities for barley phosphodiesterase. These results together with the presence of multiple species of these enzymes and a decline in net soluble RNase and DNase activities during senescence in barley leaves reported previously, make it unlikely that inhibition of RNase activity in vitro by polyamines could be correlated with their effect on senescence. Putrescine, spermidine and spermine were detected in normal and crown gall tumor tissue cultures of tobacco (Nicotiana tabacum var Wisconsin 38) and in tobacco mosaic virus (TMV)-infected freshly excised pith tissue from tobacco which represented non-proliferating tissue. The level of all three polyamines was several-fold higher in cultured tissues compared to the non-dividing freshly excised pith tissue and the tumor cultures had several-fold higher spermidine and putrescine respectively compared to normal tissue cultures. These results indicate high levels of polyamines in growing tissues but no consistent pivotal changes in polyamines during senescence. The results also do not support polyamines being natural anti-senescent compounds in plants or that their anti-senescent compounds effect could result from inhibition of RNase activity.
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PMID:Polyamine changes during senescence and tumorogenesis in plants. 369 90

DNA containing 5-azacytosine (azaC) has previously been shown to be a potent inhibitor of DNA-cytosine methyltransferases. In this report, we describe experiments which demonstrate that azaC-DNA forms a covalent complex with Hpa II methylase, a bacterial enzyme that methylates the internal C of C-C-G-G sequences. The complex does not undergo detectable dissociation over at least 3 days and is stable to denaturation with NaDodSO4. After extensive digestion of the complex with DNase and phosphodiesterase, gel filtration gave the methylase bound to approximately one equivalent of azaC; the digested complex had an apparent molecular weight similar to that of the native enzyme. Although prior treatment of azaC-DNA with Hpa II endonuclease had only a slight effect on binding of the methylase, treatment with Msp I endonuclease, which also cleaves at C-C-G-G sequences, resulted in a significant reduction in binding; this indicates that azaC residues in the recognition sequence of Hpa II are an important component in the covalent interaction of the methylase. However, since there was residual binding it is possible that azaC residues elsewhere in DNA also covalently bind to the methylase. These results provide an explanation of why azaC-DNA is such a potent inhibitor of cytosine methyltransferases and how the incorporation of such low levels of azaC into DNA can result in dramatic decreases in the methylation of cytosine. Finally, consideration of the probable catalytic mechanism of cytosine methylases and the chemical properties of azaC suggests that the inhibition is, at least in part, an active-site directed process and permits a proposal for the structure of the covalent complex.
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PMID:Covalent bond formation between a DNA-cytosine methyltransferase and DNA containing 5-azacytosine. 620 10


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