Abstract
A heat-labile alkaline phosphatase has been purified to near homogeneity from HK47, a bacterial strain isolated from Antarctic seawater. The active form of the enzyme has a molecular weight of 68,000 and is uniquely monomeric. The optimal temperature for the enzymatic activity is 25°C. Complete and irreversible thermal inactivation of the enzyme occurs in 10 min at 55°C. By using this heat-labile enzyme for dephosphorylation followed by a 10-min heat treatment, rapid end-labeling of nucleic acids by T4 polynucleotide kinase has been achieved.
Keywords: radioactive labeling, polynucleotide kinase
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- Donis-Keller H., Maxam A. M., Gilbert W. Mapping adenines, guanines, and pyrimidines in RNA. Nucleic Acids Res. 1977 Aug;4(8):2527–2538. doi: 10.1093/nar/4.8.2527. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Friedberg I., Avigad G. Some properties of alkaline phosphatase of Pseudomonas fluorescens. Eur J Biochem. 1967 Apr;1(2):193–198. doi: 10.1007/978-3-662-25813-2_30. [DOI] [PubMed] [Google Scholar]
- Heppel L. A. Selective release of enzymes from bacteria. Science. 1967 Jun 16;156(3781):1451–1455. doi: 10.1126/science.156.3781.1451. [DOI] [PubMed] [Google Scholar]
- Maxam A. M., Gilbert W. A new method for sequencing DNA. Proc Natl Acad Sci U S A. 1977 Feb;74(2):560–564. doi: 10.1073/pnas.74.2.560. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Morita R. Y. Psychrophilic bacteria. Bacteriol Rev. 1975 Jun;39(2):144–167. doi: 10.1128/br.39.2.144-167.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Richardson C. C. Phosphorylation of nucleic acid by an enzyme from T4 bacteriophage-infected Escherichia coli. Proc Natl Acad Sci U S A. 1965 Jul;54(1):158–165. doi: 10.1073/pnas.54.1.158. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schlesinger M. J., Andersen L. Multiple molecular forms of the alkaline phosphatase of Escherichia coli. Ann N Y Acad Sci. 1968 Jun 14;151(1):159–170. doi: 10.1111/j.1749-6632.1968.tb11886.x. [DOI] [PubMed] [Google Scholar]
- Schlesinger M. J., Barrett K. The reversible dissociation of the alkaline phosphatase of Escherichia coli. I. Formation and reactivation of subunits. J Biol Chem. 1965 Nov;240(11):4284–4292. [PubMed] [Google Scholar]
- Schlesinger M. J. Formation of a defective alkaline phosphatase subunit by a mutant of Escherichia coli. J Biol Chem. 1967 Apr 10;242(7):1604–1611. [PubMed] [Google Scholar]
- Schlesinger M. J. The reversible dissociation of the alkaline phosphatase of Escherichia coli. II. Properties of the subunit. J Biol Chem. 1965 Nov;240(11):4293–4298. [PubMed] [Google Scholar]
- Simoncsits A., Brownlee G. G., Brown R. S., Rubin J. R., Guilley H. New rapid gel sequencing method for RNA. Nature. 1977 Oct 27;269(5631):833–836. doi: 10.1038/269833a0. [DOI] [PubMed] [Google Scholar]
- TORRIANI A. Influence of inorganic phosphate in the formation of phosphatases by Escherichia coli. Biochim Biophys Acta. 1960 Mar 11;38:460–469. doi: 10.1016/0006-3002(60)91281-6. [DOI] [PubMed] [Google Scholar]
- Whitmore D. H., Goldberg E. Trout intestinal alkaline phosphatases. II. The effect of temperature upon enzymatic activity in vitro and in vivo. J Exp Zool. 1972 Oct;182(1):59–68. doi: 10.1002/jez.1401820107. [DOI] [PubMed] [Google Scholar]