The essential role of Ca2+ in the activity of bovine pancreatic deoxyribonuclease.
DNase requires Ca2+ for activity against DNA with Mg2+. The Ca2+ selective chelating agent, ethylene glycol bis(beta-aminoethyl ether)-N, N'-tetraacetic acid, (EGTA) inhibits DNase completely at pH 7 or 8, and subsequent addition of excess Ca2+ reverses inhibition in less than one second. DNase action can be stopped at any point by the addition of excess EGTA over Ca2+. Ca2+ is required for DNase to bind substrate. Gel filtration experiments fail to show DNase binding to 0.2 mg per ml of DNA at 5 mm Mg2+ and 10-4 M EGTA. The concentration of Ca2+ needed for half of maximum DNase activity decreases with increases DNA concentration, from 1.2 times 10-5 M Ca2+ at 2.3 times 10-5 M DNA-P to about 4 times 10-7 M Ca2+ at 2.3 DNA-P. Kinetic analysis by the titrametic assay of protons releases shows that V max is independent of Ca2+ concentration while Km increases from 7.7 times 10-5 M DNA-P at 5 times 10-4 M Ca2+ to 3.4 times 10-4 M DNA-P at 5 times 10-6 M Ca2+. Both of these results are predicted by a rate equation which is derived from the assumption that DNase must bind Ca2+ before it can bind DNA. The essential Ca2+ atom probably binds to the one of two high affinity Ca2+ binding sites on DNase which cannont bind Mg2+ or Mn2+. The only other divalent metal ions which can bind to this site, Sr2+ and Ba2+, are also the only metal ions which can substitute for Ca2+ in DNase action against DNA with Mg2+. Some DNase activity is obtained in the absence of added Ca2+ with Mg2+ at pH 6 or below and with Mn2+ or Co2+ at pH 8. These assay solutions are contaminated by 1 to 3 muM Ca2+, which may be sufficient to account for the observed activity.
D000085:Acetates / Q000494:pharmacology; D000818:Animals; D001464:Barium / Q000494:pharmacology; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology*; D002417:Cattle; D004247:DNA / Q000378:metabolism; D003851:Deoxyribonucleases / Q000378:metabolism*; D006018:Glycols / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D009691:Nucleic Acid Denaturation / Q000187:drug effects; D010179:Pancreas / Q000201:enzymology*; D011485:Protein Binding; D013324:Strontium / Q000494:pharmacology
D000085:Acetates; D006018:Glycols; D001464:Barium; D008345:Manganese; D004247:DNA; D003851:Deoxyribonucleases; D008274:Magnesium; D002118:Calcium; D013324:Strontium