Extracellular Ribonuclease Research Articles

Purification and properties of an extracellular acid ribonuclease from Penicillium janthinellum

An extracellular acid ribonuclease produced by the mold Penicillium janthinellum has been purified 26-fold by a combination of selective heat treatment, ammonium sulphate precipitation and gel filtration on Sephadex G-200. The preparation gave a single band on electrophoresis on polyacrylamide gel at three different pH values. The enzyme has a pH optimum at 3.4. It is a heat-stable enzyme having no metal requirement but it is inhibited by divalent metal ions like Cu 2+, Zn 2+ and Hg 2+. It is a protein of mol. wt 30 000 and is not an -SH enzyme. It is a phosphotransferase catalyzing the hydrolysis of ribopolynucleotides to nucleoside-3′-monophosphates, via -2′,3′-cyclic mononucleotides. When -2′,3′-cyclic mononucleotides or homopolynucleotides are used as substrates the enzyme hydrolyses those containing uracil at a much faster rate than others.

Influence of inorganic nitrate on the formation of extracellular protease and ribonuclease by Penicillium janthinellum.

The influence of nitrogen source on the formation of the extracellular acid protease and acid ribonuclease in shake cultures of the mold Penicillium janthinellum has been studied. It is observed that there is a long delay in the formation of the enzymes when nitrate is the sole source of nitrogen, but they are formed much earlier on other nitrogen sources such as ammonium salts, urea, or bacto-peptone. They are also formed earlier when nitrate is partially replaced by an ammonium salt. Small amounts of nitrite and hydroxylamine inhibit the formation of the enzymes when the mold is grown on ammonium lactate. When the mold is grown on nitrate, the culture filtrates contain inhibitory amounts of nitrite and hydroxylamine. The results indicate that the delayed formation of the enzymes in nitrate medium may be due to the formation of nitrite and hydroxylamine, which are inhibitory. The activities of the two enzymes are not inhibited by nitrite or hydroxylamine, in vitro. Prolonged preincubation of the enzymes with nitrite or hydroxylamine also does not affect the enzyme activities, indicating that it is the formation of the enzymes that is affected.

On the Reaction between the Extracellular Ribonuclease of Bacillus amyloliquefaciens (Barnase) and Its Intracellular Inhibitor (Barstar)

Abstract It is shown that the interaction between barnase and its inhibitor barstar involves the formation of a one-to-one complex which dissociates only in the presence of strongly dissociating solvents.

Observations on the Role of Inorganic Orthophosphate in the Regulation of the Formation of Bacillus amyloliquefaciens Extracellular Ribonuclease

Observations on the Role of Inorganic Orthophosphate in the Regulation of the Formation of <i>Bacillus amyloliquefaciens</i> Extracellular Ribonuclease

Production and purification of the extracellular ribonuclease of Bacillus amyloliquefaciens (barnase) and its intracellular inhibitor (barstar). I. Barnase.

Abstract Procedures are presented for the large-scale growth of Bacillus amylo-liquefaciens and the concentration and purification of its extracellular ribo-nuclease (barnase). Improvements over earlier methods include economy, the reduction of labor, and Improved purity of product, especially in the elimination of traces of protease. The bacterial cells obtained as a by-product may be used as a starting point for the isolation of barstar, the intracellular inhibitor of barnase1. Experiments relating to the purity, amino-acid composition and molecular weight of the product are also reported.

Isolation and purification of the extracellular ribonuclease from Physarum polycephalum.

The myxomycete Physarum polycephalum has been reported to produce an extracellular ribonuclease. A quick procedure is described for the isolation and purification of the ribonuclease from the culture supernatant. It involves an initial concentration of the culture supernatant by slow freezing, followed by ammonium sulfate precipitation. The enzyme was adsorbed onto DEAE-cellulose and eluted with a sodium chloride gradient.

Production and purification of the extracellular ribonuclease of Bacillus amyloliquefaciens (barnase) and its intracellular inhibitor (barstar). II. Barstar.

Abstract Procedures are reported for the extraction and purification of barstar, the intracellular inhibitor of barnase, the extracellular ribonuclease of Bacillus amyloliquefaciens, (a strain formerly erroneously classified as B. subtilis, strain H), from frozen acidified cells obtained during the isolation of barnase1. Methods include affinity chromatography on agarose to which barnase is covalently bound. Experiments relating to the purity, molecular weight, and amino acid composition of the product are reported.

Control by cesium and intermediates of the citric acid cycle of extracellular ribonuclease and other enzymes involved in the assimilation of nitrogen.

Synthesis of extracellular ribonuclease is induced in cell cultures of Ustilago sphaerogena that are starved for nitrogen and exposed to the gratuitous inducer, 6-mercaptopurine. Cesium, ammonium, or alkylammonium ion represses ribonuclease induction. Addition of citric-acid cycle intermediates to cesium ionrepressed cultures partially restores the rate of ribonuclease synthesis to the induced level. Enzymes involved in assimilation of nitrogen from different sources are also repressed by cesium ion and derepressed by intermediates from the citric acid cycle.

Purification and characterization of two extracellular ribonucleases from Rhizopus oligosporus.

Purification and characterization of two extracellular ribonucleases from Rhizopus oligosporus.

Studies on Extracellular Ribonuclease from Yeast

An extracellular ribonuclease of Rhodotorula glutinis was purified about 50-fold from the culture broth, by a procedure involving ammonium sulfate fractionation, DEAE-Sephadex column chromatography, acetone fractionation and Sephadex G-100 gel filtration. The purified enzyme was homogeneous when examined by the ultracentrifuge and disc electrophoresis. The molecular weight was calculated to be 58,900 and 56,000 by the sedimentation-diffusion method and the sedimentation equilibrium method, respectively.

Studies on Extracellular Ribonuclease from Yeast

Studies on Extracellular Ribonuclease from Yeast

Role of extracellular ribonuclease in growth of Corynebacterium acnes.

Corynebacterium acnes produces an extracellular nuclease which degrades ribonucleic acid. Growth was stimulated in peptone medium without glucose by the addition of ribonucleic acid and was related to the amount of ribonucleic acid depolymerized. Among other bacteria examined nuclease production was not always associated with the stimulation of growth by ribonucleic acid. Growth of C. acnes in peptone broth was stimulated by xanthine and guanine. Growth was not initiated in a basal nucleic acid free (NAF) medium by the addition of several nucleic acid metabolites nor in the NAF basal medium containing only 0.05% yeast extract. Growth occurred when the NAF–yeast extract base was supplemented with ribonucleic acid or other nucleic acid metabolites. Ribose and ribonucleic acid were equal in growth-supporting ability. Some purine bases in combination with glucose or ribose also supported growth. Other bases such as adenine or cytosine were not stimulatory but their ribotides were. Ribonuclease functioned as a growth stimulating enzyme by its action on ribonucleic acid under certain cultural conditions. Ribose appears to be a major stimulating metabolite made available from nucleic acid breakdown. Other components such as ribotides or nucleic acid bases, particularly purines, will also support growth in media devoid of these compounds or lacking them in sufficient concentration.

Extracellular nuclease activity of fish spoilage bacteria, fish pathogens, and related species.

The production of extracellular deoxyribonuclease and ribonuclease by 23 marine and 3 dairy strains of Pseudomonas putrefaciens, 15 strains of fish-pathogenic fluorescent pseudomonads, 38 strains of fluorescent pseudomonads isolated from haddock, and 34 related organisms was determined by an agar plate method. All strains of P. putrefaciens produced both deoxyribonuclease and ribonuclease. Of the other 87 organisms examined, 26.5% produced ribonuclease and 14.5% produced deoxyribonuclease. All organisms which produced deoxyribonuclease also produced ribonuclease. Deoxyribonuclease production by P. putrefaciens is suggested as a useful criterion of identity for members of this intense fish spoilage species.

Preferential synthesis of extracellular ribonuclease and some intracellular proteins in actinomycin D-treated Bacillus subtilis

1. Actinomycin D, at a concentration of 1 μg/ml, inhibited almost completely the formation of extracellular α-amylase (EC 3.2.1.1) by Bacillus subtilis K, while the accumulation of ribonuclease (EC class 2.7.7) continued at a decreased, but constant, rate in the same culture. The latter was, however, inhibited immediately and completely by the addition of 5 μg/ml of the antibiotic. 2. Actinomycin D (1 μg/ml) did not completely inhibit the incorporation of labeled leucine into cellular proteins; a decreased rate of incorporation (10–20 % of the control) remained for at least 60 min in the actinomycin D-treated cells. On the other hand, 5 μg/ml of this drug resulted in the inhibition of leucine incorporation (more than 97 % inhibition) within 30 min. 3. Purification of individual enzymes demonstrated that the actinomycin D-treated cells incorporated labeled leucine well into the ribonuclease protein but insignificantly into the α-amylase protein. The inhibition ratio, as judged by the enzyme formation, was in good agreement with that calculated from the leucine incorporation. 4. Fractionation by DEAE-cellulose column chromatography and gel filtration on Sephadex G-75 indicated that labeled leucine was also incorporated preferentially into limited species of intracellular proteins in the presence of 1 μg/ml of actinomycin D, as in the case of the syntheses of extracellular enzymes. The results clearly indicate that actinomycin D at low concentrations has differential effects upon the syntheses de novo of intracellular proteins, as well as extracellular enzymes in B. subtilis K cells.

Extracellular Nuclease Activity of Fish Spoilage Bacteria, Fish Pathogens, and Related Species

The production of extracellular deoxyribonuclease and ribonuclease by 23 marine and 3 dairy strains of Pseudomonas putrefaciens, 15 strains of fish-pathogenic fluorescent pseudomonads, 38 strains of fluorescent pseudomonads isolated from haddock, and 34 related organisms was determined by an agar plate method. All strains of P. putrefaciens produced both deoxyribonuclease and ribonuclease. Of the other 87 organisms examined, 26.5% produced ribonuclease and 14.5% produced deoxyribonuclease. All organisms which produced deoxyribonuclease also produced ribonuclease. Deoxyribonuclease production by P. putrefaciens is suggested as a useful criterion of identity for members of this intense fish spoilage species.

A reversible thermal transition of the extracellular ribonuclease of Bacillus amyloliquefaciens.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTA reversible thermal transition of the extracellular ribonuclease of Bacillus amyloliquefaciensRobert W. HartleyCite this: Biochemistry 1968, 7, 6, 2401–2408Publication Date (Print):June 1, 1968Publication History Published online1 May 2002Published inissue 1 June 1968https://doi.org/10.1021/bi00846a050RIGHTS & PERMISSIONSArticle Views68Altmetric-Citations38LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (723 KB) Get e-Alerts Get e-Alerts

Studies on extracellular ribonucleases of Ustilago sphaerogena. Characterization of substrate specificity with special reference to purine-specific ribonucleases.

1. Ribonuclease U(1) splits only the phosphodiester bonds of guanosine 3'-phosphates in RNA. It may be regarded as a guanyloribonuclease [ribonucleate (guanine nucleotide)-2'-transferase (cyclizing), EC 2.7.7.26] similar to ribonuclease T(1) (Egami, Takahashi & Uchida, 1964). It seems to be identical with the extracellular ribonuclease described by Glitz & Dekker (1963, 1964a,b). 2. Ribonucleases U(2) and U(3) are novel enzymes with a strict specificity. They split the internucleotide bonds between purine 3'-nucleotides and 5'-hydroxy groups of adjacent nucleotides in RNA with the intermediary formation of purine nucleoside 2',3'-(cyclic)-phosphates, which are slowly hydrolysed to purine 3'-nucleotides. So they may be classified as ;puryloribonucleases [ribonucleate (purine nucleotide)-2'-transferase (cyclizing)]'. Double-stranded RNA is scarcely split by ribonucleases U(2) and U(3). 3. Ribonuclease U(4) has no absolute base specificity, and produces the mononucleotides 3'-adenylate, 3'-guanylate, 3'-cytidylate and 3'-uridylate from RNA.

Studies on extracellular ribonucleases of Ustilago sphaerogena. Purification and properties.

1. Four ribonucleases were isolated from culture media of Ustilago sphaerogena. They were designated ribonucleases U(1), U(2), U(3) and U(4). 2. They were purified about 1600-, 3700-, 1100- and 16-fold respectively. 3. It was shown by gel filtration that ribonucleases U(1), U(2) and U(3) have molecular weights about 10000 like ribonuclease T(1), and that ribonuclease U(4) is much larger. 4. Ribonucleases U(1), U(2) and U(3) are thermostable, but ribonuclease U(4) is not. 5. The pH optimum of ribonucleases U(1) and U(4) is pH8.0-8.5, and that of ribonucleases U(2) and U(3) is pH4.5.

Isolation and properties of a specific bacterial ribonuclease inhibitor

1. 1. Cultures of Bacillus subtilis cells produce extracellular ribonuclease towards the end of the growth phase. At the same time there is the production inside the cell of an inhibitor which is capable of inactivating the extracellular enzyme. 2. 2. The inhibitor can be selectively released by suddenly cooling the cells over a 14–16° temperature range. Such cold shocked cells lose only 4 % of total protein and show no gross morphological changes. Protoplast studies show the inhibitor to occur inside the cell membrane. 3. 3. The inhibitor has been purified and shown to have the properties of a protein of mol. wt. 12 500. Its properties are described. 4. 4. The inhibitor apparently forms a complex with the enzyme; inhibiton is not reversible by urea, salt, heating or sulphydryl reagents in contrast to the mammalian system. It has no action on pancreatic ribonuclease or ribonuclease T 1. 5. 5. Using the paradoxical effect of actinomycin in stimulating ribonuclease synthesis in B. subtilis it has been shown that inhibitor and ribonuclease are independently synthesised and are not parts of a primary zymogen molecule. The possibility that the inhibitor is a secretory carrier for the enzyme has also been excluded.

Ribonuclease activity in the Ehrlich ascites tumour.

PREVIOUS studies1 have revealed a relationship between the distribution of intracellular and extracellular ribonucleases and the phase of growth of Staphylococcus aureus strain Duncan. The difficulty of distinguishing between intracellular and extracellular enzymes precludes similar studies on animal tissues, but it was considered desirable to examine such enzymes in the ascites tumour, where separation of cells and ascitic fluid presents no difficulty.