Motoaki Anai

Degradation of deoxyribonucleic acid by guinea pig epidermal extracts.

SummaryThe capability of guinea pig epidermal extracts to hydrolyze deoxyribonucleic acid has been studied. The results of investigation by gel filtration on Sephadex G-75 column and by viscometry on the mode of hydrolysis of deoxyribonucleic acid by epidermal extracts revealed that deoxyribonucleic acid was degraded by both endonuclease and exonuclease activities. The exhaustive digestion by epidermal extracts yielded the complete degradation of deoxyribonucleic acid to mononucleosides or further metabolites. The enzyme systems involved in the deoxyribonucleic acid degradation include at least an endonuclease, an exonuclease and a phosphatase.ZusammenfassungEine Untersuchung über die DNS-Hydrolyse durch Meerschweinchen-Epidermis-Extrakt wurde durchgeführt. Gel-Filtration in der Sephadex G-75 Säule und Viskometrie ergaben eine DNS-Zerstörung durch Endonuclease- wie auch der Exonuclease-Aktivitäten. Die Verdauung zeigt eine völlige DNS-Zerstörung bis zu Mononucleosiden oder weitergehenden Metaboliten. Unter den Enzymen der DNS-Verdauung spielen zumindest eine Endonuclease, eine Exonuclease und eine Phosphatase eine Rolle.

Purification and characterization of guinea-pig epidermal acid phosphatase.

Guinea‐pig epidermal acid phosphatase has been purified approximately 120‐fold by a procedure including acid treatment, CM‐cellulose and DEAE‐cellulose chromatography, and gel filtration on Sephadex G‐100. The enzyme had a pH optimum at 5.0 and the optimal temperature for activity was approximately 50 C. The enzyme was not activated by divalent cations or 2‐mercaptoethanol, but it was inhibited by p‐chloromercuribenzoate and by fluoride. The km value for p‐nitrophenyl phosphate was 1.31 × 10−4 M, the molecular weight was about 73,000 as determined by Sephadex G‐100 gel filtration and the isoelectric point was 6.1 The enzyme hydrolyzed deoxyribonucleoside monophosphates to deoxyribonucleosides.

Endodeoxyribonuclease from nuclei of bovine small intestinal mucosa: Further studies on intranuclear localization and cleavage mechanism of the enzyme

Most of the activity of endodeoxyribonuclease was extracted from isolated chromatin with buffer containing 0.6 M NaCl, indicating that the endonuclease is present as a chromatin-bound form. When nuclei or chromatin of calf thymus or rat liver was digested with the bovine nuclear enzyme in the presence of 2 mM EGTA, to suppress endogenous Ca, Mg-dependent DNase activity, discrete DNA bands with integral multiples of 200 base pairs in length were produced, but no acid-soluble nucleotide was detected. The enzyme made single-strand breaks in pBR322 DNA and degraded it to fragments of limited size. The size of the final products of DNAs of Micrococcus luteus, rat liver nuclei, and calf thymus nuclei was about 3,000, 200, and 160 base pairs, respectively, but the enzyme showed no base specificity. Thus the endonuclease seems preferentially to recognize AT-rich regions of double-stranded DNA and to make single-strand breaks.

Properties of the RecB and RecC gene products of EscherichiaColi

The properties of the recB and recC gene products of Escherichia coli were studied using recB and recC gene-inserted plasmids. recB mutants and recC mutants lacked ATP-dependent DNase (recBC enzyme) but showed apparent recovery of enzyme activity on introduction of plasmids carrying the recB and recC gene, respectively. The ATP-dependent DNase was also constructed in vitro by mixing the recB and recC gene products encoded by the plasmids with the corresponding gene. Specific labeling of plasmid-encoded proteins by the maxicell method showed that the recB and recC gene products were 135,000 and 125,000 dalton proteins, respectively. These results suggest that the recB and recC genes are the structural genes of the beta and alpha subunits, respectively, of the recBC enzyme. A gene that encodes a protein of about 100,000 daltons was found to be located between the recB and recC genes. But the product of this gene was shown not to be included in the recBC enzyme.