Assay method for femtogram order of Ca2+, Mg(2+)-dependent endonuclease.
A sensitive method for the assay of Ca2+, Mg(2+)-dependent endonuclease was developed. The assay procedure is composed of two parts: (i) microscale endonuclease digestion of highly polymerized calf thymus DNA and (ii) the quantification of DNA breaks by measuring the activation of poly(ADP-ribose) polymerase, which is known to be activated proportionally to the number of nicks and ends of DNA added in the reaction mixture. This method was approximately 10(5)-fold more sensitive than a conventional DNase assay detecting acid-soluble DNA formation and, thus, the activity of 20 to 100 fg of purified bull seminal Ca2+, Mg(2+)-dependent endonuclease could be reliably measured. Ca2+ and Mg2+ requirements and the response to histone H2B of the endonuclease were also demonstrated by this method. Using this method, the assay of a very small amounts of Ca2+, Mg(2+)-dependent endonuclease in crude extracts of calf thymus chromatin was possible. This method may be applied to other types of endonucleases by modifying the mixture for endonuclease reaction.
Yoshihara
K
K
Department of Biochemistry, Nara Medical University, Japan.
Ohta
I
I
Tanaka
Y
Y
Fujita
H
H
Ide
T
T
Ohgushi
H
H
D000818:Animals; D002417:Cattle; D002843:Chromatin / Q000201:enzymology; D004247:DNA; D004706:Endodeoxyribonucleases / Q000032:analysis*; D004789:Enzyme Activation; D005069:Evaluation Studies as Topic; D066298:In Vitro Techniques; D008297:Male; D008832:Microchemistry; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D012661:Semen / Q000201:enzymology; D012680:Sensitivity and Specificity; D013379:Substrate Specificity; D013950:Thymus Gland / Q000201:enzymology
D002843:Chromatin; D004247:DNA; D011065:Poly(ADP-ribose) Polymerases; D004706:Endodeoxyribonucleases; C029367:calcium magnesium dependent endodeoxyribonuclease