Extracellular nucleases of Bacillus subtilis I. Purification and properties

Kanamori, Norio; Sakabe, K.; Okazaki, Reiji

doi:10.1016/0005-2787(74)90220-2

Cited by 33 publications

(38 citation statements)

References 18 publications

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“…This step also helps in the removal of the endogenous proteolytic activity present in the seed extracts [32]. About 50 % of the proteins present in the crude extract were removed by this step.…”

Section: Discussionmentioning

confidence: 99%

“…Anionic inhibitors have been examined by the Davis method [43], cationic inhibitors by the Reisfeld method [32] and neutral inhibitors by Weber and Osborn [44] and Laemmli [45] methods. While these methods have proved useful for establishing the purity and determining the molecular weight of proteins, they cannot be used to distinguish isoinhibitors or compare the activity of the particular inhibitor against different proteolytic enzymes.…”

Section: Discussionmentioning

confidence: 99%

“…Polyacrylamide gel electrophoresis was carried out as described by Reisfield et al, [32] and followed by Gabriel [33]. PAGE was carried out under non-denaturing condition using 12 % slab gels.…”

Section: Extraction and Purification Of Sntismentioning

confidence: 99%

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Evaluating the role of a trypsin inhibitor from soap nut (Sapindus trifoliatus L. Var. Emarginatus) seeds against larval gut proteases, its purification and characterization

et al. 2015

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Evaluating the role of a trypsin inhibitor from soap nut (Sapindus trifoliatus L. Var. Emarginatus) seeds against larval gut proteases, its purification and characterization

et al. 2015

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“…The DNase purified by Kerr et al [14,15] is not an intracellular enzyme but is excreted in the medium and it is an exonuclease, producing 3'-mononucleotides. The different DNases purified by Okazaki and his collaborators [16-181 are all extracellular enzymes, are not specific for single-stranded DNA (except for the BS-I0 enzyme [17]) and have all an exonucleolytic mode of action. Birnboim [19] described a CaZ+-activated DNase in B. suhtilis specific for singlestranded DNA; the enzyme, on the other hand, has a pH optimum higher than 9.0 (whereas the activity of our enzyme decreases beyond pH 8) and its mode of action is exonucleolytic.…”

Section: Discussionmentioning

confidence: 99%

An Intracellular Endonuclease of Bacillus subtilis Specific for Single‐Stranded DNA

Ciarrocchi¹,

Fortunato²,

Cobianchi³

et al. 1976

European Journal of Biochemistry

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We have fractionated from extracts of Bacillus subtilis the DNase activity specific for singlestranded DNA; the activity separates in two main fractions on Sephadex G-200, a larger one ( M , > 400000) and a smaller one ( M , z 30000). We have purified the smaller, more abundant fraction nearly 3000-fold. The purified enzyme has a pH optimum close to 8, is activated by Ca", and is inhibited by EDTA ; the enzyme hydrolyses single-stranded DNA at a rate approximately 40 times greater than double-stranded DNA. The mode of action is endonucleolytic on both substrates, but the possibility that the two activities may reside on different molecules is not ruled out. The products have 5'-P and 3'-OH ends.The enzyme is different from those purified from the culture media of the same organism in several respects; the latter are all extracellular enzymes, they are not specific for single-stranded DNA (except one) and have all an exonucleolytic mode of action.A description of the properties of the deoxyribonucleases of Bacillus subtilis is essential to understand the pathways of genetic recombination in this organism. Chestukhin et al. [l], Doly and Anagnostopoulos [2] and Ohi and Sueoka [3] have described an ATPdependent DNase from this organism; two mutants, belonging to two different genes have been described which are reduced in the level of that enzyme [1,2] and are deficient in recombination and in several repair properties, in analogy with the comparable mutants of Diplococcuspneumoniae [4] and Escherichia coli [5]. No other enzyme of Bacillus subtilis has been associated with the recombination process.In our laboratory we have isolated a number of mutants of Bacillus subtilis deficient in recombination and/or in several DNA repair properties [6-81. A characterization of the levels of some enzymes in these mutants led to the identification of a polA mutant [6,9] and to the observation of a partial defect in an intracellular DNase acting on singlestranded DNA in another mutant named rec-30 [XI.We have thus begun a study of the intracellular nucleases hydrolyzing single-stranded DNA. We report here the initial results of our work, describing one of the enzymes which catalyze this reaction. Enzymes. Pancreatic DNase (EC 3

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“…This nuclease has a pH optimum of 9.5 for exonucleolytic degradation of RNA to nucleoside 3Ј-monophosphates. Apparently the same nuclease (Bs-II) and two others (Bs-IA and Bs-IB) were partially purified later by Kanamori et al (103,104). All three were Ca 2ϩ dependent and had both RNase and DNase activity, leading to the production of nucleoside 3Ј-monophosphates.…”

Section: Rnases For Which Genes Are Not Yet Identifiedmentioning

confidence: 97%

RNA Processing and Degradation inBacillus subtilis

Condon

2003

Microbiol Mol Biol Rev

141

134

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This review focuses on the enzymes and pathways of RNA processing and degradation in Bacillus subtilis, and compares them to those of its gram-negative counterpart, Escherichia coli. A comparison of the genomes from the two organisms reveals that B. subtilis has a very different selection of RNases available for RNA maturation. Of 17 characterized ribonuclease activities thus far identified in E. coli and B. subtilis, only 6 are shared, 3 exoribonucleases and 3 endoribonucleases. Some enzymes essential for cell viability in E. coli, such as RNase E and oligoribonuclease, do not have homologs in B. subtilis, and of those enzymes in common, some combinations are essential in one organism but not in the other. The degradation pathways and transcript half-lives have been examined to various degrees for a dozen or so B. subtilis mRNAs. The determinants of mRNA stability have been characterized for a number of these and point to a fundamentally different process in the initiation of mRNA decay. While RNase E binds to the 5′ end and catalyzes the rate-limiting cleavage of the majority of E. coli RNAs by looping to internal sites, the equivalent nuclease in B. subtilis, although not yet identified, is predicted to scan or track from the 5′ end. RNase E can also access cleavage sites directly, albeit less efficiently, while the enzyme responsible for initiating the decay of B. subtilis mRNAs appears incapable of direct entry. Thus, unlike E. coli, RNAs possessing stable secondary structures or sites for protein or ribosome binding near the 5′ end can have very long half-lives even if the RNA is not protected by translation

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Extracellular nucleases of Bacillus subtilis I. Purification and properties

Cited by 33 publications

References 18 publications

Evaluating the role of a trypsin inhibitor from soap nut (Sapindus trifoliatus L. Var. Emarginatus) seeds against larval gut proteases, its purification and characterization

Evaluating the role of a trypsin inhibitor from soap nut (Sapindus trifoliatus L. Var. Emarginatus) seeds against larval gut proteases, its purification and characterization

An Intracellular Endonuclease of Bacillus subtilis Specific for Single‐Stranded DNA

RNA Processing and Degradation inBacillus subtilis

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