Purification and characterization of the potent endonuclease in extracts of bovine heart mitochondria.

Oscar Cummings, T. C. King, J. A. Holden, R. L. Low

Research output: Contribution to journalArticle

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Abstract

A potent endonuclease identified in a crude fraction of soluble proteins from bovine heart mitochondria has been purified 2500-fold and partially characterized. Physical studies of the enzyme indicate a Stokes radius of 30.3 A and a sedimentation coefficient, S20 degrees, w, of 4.1 yielding a native molecule weight of 59,000 and a frictional coefficient of 1.2. Analysis of extensively purified fractions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals a major band at 29,000 Da accounting for 50% of the total protein and suggesting a dimeric subunit structure. The endonuclease maintains two distinct pH optima: pH 5.1-5.5 and 7-8. Both acid and neutral activities nick supercoiled M13 circular double-stranded replicative form I DNA and fragment single-stranded DNA templates to generate 5'-phosphoryl-3'-hydroxyl breaks. The endonuclease requires a divalent cation (preferring Mn2+ over Mg2+) and is sensitive to N-ethylmaleimide and moderate levels of salt. Analysis of the digestion products of double-stranded DNA after prolonged nuclease treatment yields a mixture of oligonucleotides, 13% of which are di- and trinucleotides. Despite the enzyme's ability to degrade DNA to oligonucleotides under some conditions, a strikingly nonrandom pattern of cleavage is observed when a restriction fragment composed of bovine D-loop DNA is used as a template. In this case, a strong preference for guanine tracts is seen.

Original languageEnglish
Pages (from-to)2005-2015
Number of pages11
JournalJournal of Biological Chemistry
Volume262
Issue number5
StatePublished - Feb 15 1987
Externally publishedYes

Fingerprint

Heart Mitochondria
Mitochondria
Endonucleases
Purification
DNA
Oligonucleotides
Ethylmaleimide
Single-Stranded DNA
Divalent Cations
Guanine
Enzymes
Electrophoresis
Sedimentation
Sodium Dodecyl Sulfate
Hydroxyl Radical
Polyacrylamide Gel Electrophoresis
Digestion
Proteins
Salts
Weights and Measures

ASJC Scopus subject areas

  • Biochemistry

Cite this

Purification and characterization of the potent endonuclease in extracts of bovine heart mitochondria. / Cummings, Oscar; King, T. C.; Holden, J. A.; Low, R. L.

In: Journal of Biological Chemistry, Vol. 262, No. 5, 15.02.1987, p. 2005-2015.

Research output: Contribution to journalArticle

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