Biomedical subjects

M Anai

Publications and source records attributed to M Anai.

At least 91 records · Page 5Linked to original sources

PubMed1980

Purification and properties of deoxyribonuclease II from human urine.

The acid deoxyribonucleases [DNase II; EC 3.1.4.6] in human urine were purified approximately 400- to 500-fold by phosphocellulose chromatography, gel filtration on Sephadex G-75 and isoelectric focusing, with a total recovery of 22%. The enzymes were present in a least three forms with different isoelectric points, pHs 6.4, 6.6, and 6.8. However, other properties were essentially similar. The enzymes did not require divalent cations for activity, and the optimal pHs were at 5.1 to 5.3 in 33 mM acetate buffer. They had a molecular weight of around 36,000, as estimated by gel filtration on Sephadex G-75. The enzymes were endonucleases which hydrolyzed native, double-stranded DNA about 5 to 15 times faster than thermally denatured DNA. The products formed from native DNA were 3'-phosphoryl- and 5'-hydroxy-terminated oligonucleotides. The average chain length of the limit digests with these enzymes was approximately 11 to 15, and the major fragments were longer than pentanucleotides. The final preparations were free of nonspecific acid and alkaline phosphatases and phosphodiesterase, but contained contaminating ribonuclease activity.

Deoxyribonucleases↗

PubMed1979

Inhibition of the recBC enzyme of Escherichia coli by specific binding of pyridoxal 5'-phosphate to DNA binding site.

Pyridoxal 5'-phosphate rapidly abolished the DNA-hydrolyzing activities as well as DNA-dependent ATP-ase activity of the recBC enzyme of Escherichia coli. Pyridoxal also had an inhibitory effect on the enzyme but less effective than that of pyridoxal 5'-phosphate. Pyridoxamine 5'-phosphate, pyridoxamine, or pyridoxine had no effect on the activities of the enzyme. The inhibition was rapidly reversed by dilution but could be made irreversible by reduction with sodium borohydride prior to dilution. This suggests the formation of Schiff base between pyridoxal 5'-phosphate and an epsilon-amino group of a lysine residue which is essential for the enzyme activity. Pyridoxal 5'-phosphate is a competitive inhibitor of DNA substrate but not of ATP. Furthermore, the presence of DNA substrate protected the enzyme from inactivation by the reduction but the presence of ATP showed no effect. Thus, the recBC enzyme appears to have an essential lysine residue at or near the DNA binding site of the enzyme, and the enzyme possesses two independent catalytic sites, such as a DNA binding site and an ATP binding site.

DNA, Bacterial↗

PubMed1978

Purification and properties of deoxyribonuclease from human urine.

The DNAase in human urine was purified about 30-fold with a recovery of 28%. This involved DEAE-cellulose and phosphocellulose chromatography steps and gel filtration on Sephadex G-75. The enzyme required divalent cations such as Co2+, Mg2+, Mn2+ and Zn2+ for activity, but Ca2+, Cu2+ and Fe2+ were ineffective. EDTA and G-actin inhibited the reaction. The maximum activity was observed at pH 5.5 in acetate buffer plus Co2+ or Mg2+ and Ca2+. It had a molecular weight of approximately 38 000, estimated by gel filtration on Sephadex G-75 and isoelectric point of around pH 3.9. The enzyme is an endonuclease which hydrolyzes native, double-stranded DNA about 3 to 4 times faster than thermally denatured DNA to produce 5'-phosphoryl- and 3'-hydroxyl-terminated oligonucleotides. The final preparation was free of non-specific acid and alkaline phosphatases, phosphodiesterase and ribonuclease activities.

Chromatography, Gel↗

PubMed1977

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 degrees 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 1-31x10-4 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.

Acid Phosphatase↗

PubMed1975

Degradation of deoxyribonucleic acid by guinea pig epidermal extracts.

The 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.

Animals↗

PubMed1975

A nucleoside triphosphate-dependent deoxyribonuclease from Bacillus laterosporus. The mode of action of the enzyme.

The acid-soluble products of exhaustive digestion of native DNA with Bacillus laterosporus DNase consist of 6.5% of mononucleotides and 93.5% of oligonucleotides with an average chain length of 3.2. The results of viscometric studies and inactivation of transforming DNA indicate the existence of acid-insoluble intermediates and the selective degradation of the population of substrate molecules rather than a random nucleolytic action. Furthermore, sucrose density gradient analysis of partially digested DNA showed that the initial DNA added as a substrate disappeared progressively during the reaction, being replaced by much more slowly sedimenting acid-insoluble materials, which were eventually degraded into acid-soluble end products during the reaction; products intermediate in size between these two components were not detectable. Studies with DNA labeled at the 3'-terminus indicate that Bacillus laterosporus DNase does not attack DNA from 3'-hydroxyl ends to yeild acid-soluble or acid-insoluble materials in a random manner. The results presented in this paper indicate that the nature of the attack of B. laterosporus nuclease is similar to that previously proposed for Micrococcus luteus DNase. The possibility of the sequential release of acid-insoluble intermediate fragments as well as acid-soluble products from the terminal portion of DNA by the enzyme is discussed.

Bacillus↗

PubMed1975

A nucleoside triphosphate-dependent deoxyribonuclease from Bacillus laterosporus. Purification and characterization of the enzyme.

A deoxyribonuclease, which requires nucleoside triphosphate for reaction, has been purified about 150-fold from extracts of Bacillus laterosporus. Potassium phosphate and ethylene glycol stabilize the purified enzyme. The enzyme degrades double-stranded DNA about 100 times faster than heat-denatured DNA in the presence of nucleoside triphosphate. Double-stranded DNA is not degraded to any measurable extent in the absence of ATP, but the enzyme exhibits activity toward denatured DNA in the absence of nucleoside triphosphate, and this activity seems to be an intrinsic property of this enzyme protein. The optimum pH is 8.5 and the maximum activity is obtained in the copresence of Mg2+ (8.0 X 10(-3)M) and Mn2+ (7.0 X 10(-5)M). ATP and dATP are most effective and nucleoside di- or monophosphates are ineffective. ATP is converted to ADP and inorganic phosphate during the reaction and the ratio of the amount of ATP cleaved to that of hydrolyzed phosphodiester bonds of DNA is about 3:1. An inhibitor of the enzyme was observed in bacterial extracts prepared by sonic disruption; the inhibitory substance is produced in the bacteria in the later stages of cell growth. Preliminary results show that the inhibitor emerged near the void volume of a Sephadex G-200 column, and was relatively heat-stable, RNase-resistant, and DNase-sensitive.

Adenosine Triphosphate↗

PubMed1974