Purification and properties of the major nuclease from mitochondria of Saccharomyces cerevisiae

The vast majority of nuclease activity in yeast mitochondria is due to a single polypeptide with an apparent molecular weight of 38,000. The enzyme is located in the mitochondrial inner membrane and requires non-ionic detergents for solubilization and activity. A combination of heparin-agarose and C...

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Bibliographic Details
Published in:The Journal of biological chemistry 1988-06, Vol.263 (16), p.7691-7702
Main Authors: Dake, E, Hofmann, T J, McIntire, S, Hudson, A, Zassenhaus, H P
Format: Article
Language:English
Subjects:
Analytical, structural and metabolic biochemistry
Biological and medical sciences
Cations, Divalent - analysis
Deoxyribonucleases - isolation & purification
Enzymes and enzyme inhibitors
EPURATION
Ethidium - pharmacology
Fundamental and applied biological sciences. Psychology
Hydrogen-Ion Concentration
MITOCHONDRIA
Mitochondria - enzymology
MITOCHONDRIE
MITOCONDRIA
Molecular Weight
NUCLEOSIDASAS
NUCLEOSIDASE
NUCLEOSIDASES
PURIFICACION
PURIFICATION
Ribonucleases - isolation & purification
SACCHAROMYCES CEREVISIAE
Saccharomyces cerevisiae - enzymology
Saccharomyces cerevisiae - ultrastructure
Spermidine - pharmacology
Temperature
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Summary:The vast majority of nuclease activity in yeast mitochondria is due to a single polypeptide with an apparent molecular weight of 38,000. The enzyme is located in the mitochondrial inner membrane and requires non-ionic detergents for solubilization and activity. A combination of heparin-agarose and Cibacron blue-agarose chromatography was employed to purify the nuclease to approximately 90% homogeneity. The purified enzyme shows multiple activities: 1) RNase activity on single-stranded, but not double-stranded RNA, 2) endonuclease activity on single- and double-stranded DNA, and 3) a 5‘-exonuclease activity on double-stranded DNA. Digestion products with DNA contain 5‘-phosphorylated termini. Antibody raised against an analogous enzyme purified from Neurospora crassa (Chow, T. Y. K., and Fraser, M. (1983) J. Biol. Chem. 258, 12010-12018) inhibits and immunoprecipitates the yeast enzyme. This antibody inhibits 90-95% of all nuclease activity present in solubilized mitochondria, indicating that the purified nuclease accounts for the bulk of mitochondrial nucleolytic activity. Analysis of a mutant strain in which the gene for the nuclease has been disrupted supports this conclusion and shows that all detectable DNase activity and most nonspecific RNase activity in the mitochondria is due to this single enzyme.
ISSN:0021-9258
1083-351X
DOI:10.1016/S0021-9258(18)68554-0