Age-dependent conformational changes in acid deoxyribonuclease of chick brain

Abstract

Acid deoxyribonuclease (EC 3.1.4.6) (DNase) from young (16 days of incubation) and old (1.5 years) chick cerebral hemispheres was purified to apparent homogeneity. Throughout the purification schedule, the behavior of “young” and “old” enzymes was similar. However, the specific activity of the purified enzyme from old brain was only one-tenth that of young enzyme. Polyacrylamide gel electrophoresis of the purified acid DNase gave a single band. Antisera against both “young” and “old” enzyme were raised and double immunodiffusion experiments revealed cross-reaction of young antigen with old antiserum and vice versa, although precipitin bands with young antigen against young antiserum and old antigen against old antiserum were more sharp. Both young and old acid DNase preparations showed an apparent molecular weight of 62 000 and many other properties like heat stability, effect of various exogenous compounds like Hg 2+, Zn 2+, Mg 2+, etc., were also similar. The old enzyme showed slightly higher K m and decreased V max compared with the young enzyme. Dansylation of N-terminal amino acids and their analysis following tryptic digestion of both “young” and “old” acid DNase revealed a similar pattern. Immunotitration experiments showed that the old enzyme requires more antiserum prepared against “young” enzyme to achieve 50% inactivation, thus pointing out the presence of completely or partially inactive molecules in “old” acid DNase preparation. Circular dichroism spectra of the enzyme preparations indicated that the “old” acid DNase molecules are more rigid and have more δ-helical structure, compared with the “young” enzyme. From these data, it is suggested that the reduction in the specific activity of old acid DNase may be, apart from other possibilities, due to conformational changes in the enzyme molecules.