Mechanism for inhibition of deoxyribonuclease activity by antisera.
The activity of bovine DNase, but not that of porcine DNase, is inhibited by antisera against bovine DNase, and vice versa. Inhibition of DNase is found in the immunoglobulin G-containing fractions, as shown by ion exchange chromatography. Inactive DNase, carboxymethylated specifically at the active site His134, competes with active DNase and reverses the antisera inhibition of DNase, suggesting that the epitode responsible for inhibition does not contain the active site His134. Alignment of the sequences of DNase of these two species shows that the greatest variation occurs between residues 153 and 163, within which are three consecutive peptide bonds, Lys-Trp-His-Leu, that are readily cleaved by trypsin, chymotrypsin, or thermolysin. The 8-hr digest of DNase by each of these three proteases has lost the ability to reverse antisera inhibition. The degree of antisera inhibition varies with the metal ion used as the activator for DNase-catalyzed reactions. When Mn2+, Co2+, or Mg2+ plus Ca2+ are used as activators, inhibition is approximately 50%. When pBR322 plasmid is used as substrate, gel electrophoresis shows that the DNase-catalyzed DNA hydrolysis produces a significant amount of double-strand cuts with Mn2+, Co2+, or Mg2+ plus Ca2+ as activators and antisera inhibit DNase action only on double-strand cuts. With only Mg2+ as the activator no double-strand cuts are observed, either in the presence or absence of antisera, and the DNase activity is not significantly inhibited. We conclude that antisera inhibition is due to antibody binding of the DNase polypeptide chain within residues 153 and 163. These residues are not crucial for catalysis, but are required for DNA binding, which results in double-strand cuts.
Liu
Y F
YF
Institute of Biochemistry, College of Medicine, National Taiwan University, Taipei.
Liao
T H
TH
D000818:Animals; D000918:Antibody Specificity; D001665:Binding Sites; D001666:Binding Sites, Antibody; D002118:Calcium / Q000494:pharmacology; D002417:Cattle; D003035:Cobalt / Q000494:pharmacology; D004278:DNA, Superhelical / Q000378:metabolism; D003851:Deoxyribonucleases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology; D006639:Histidine; D007106:Immune Sera / Q000494:pharmacology*; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008670:Metals / Q000494:pharmacology; D008958:Models, Molecular; D013552:Swine
D004278:DNA, Superhelical; D000939:Epitopes; D007106:Immune Sera; D008670:Metals; D003035:Cobalt; D008345:Manganese; D006639:Histidine; D003851:Deoxyribonucleases; D008274:Magnesium; D002118:Calcium