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Age-dependent conformational changes in acid deoxyribonuclease of chick brain
Insights
The specific activity of acid deoxyribonuclease (DNase) in old chick brains is ten times lower than in young brains. This reduction is linked to conformational changes and increased rigidity in the older DNase molecules.
Area of Science:
- Biochemistry
- Molecular Biology
- Neuroscience
Background:
- Acid deoxyribonuclease (DNase) is an enzyme found in chick cerebral hemispheres.
- Enzyme activity can change with age, impacting cellular function.
- Understanding age-related changes in enzyme properties is crucial for studying aging processes.
Purpose of the Study:
- To investigate the differences in acid DNase from young and old chick cerebral hemispheres.
- To determine the cause of reduced specific activity in older acid DNase.
- To characterize the structural and functional properties of young and old acid DNase.
Main Methods:
- Purification of acid DNase from young and old chick brains.
- Polyacrylamide gel electrophoresis (PAGE) for homogeneity assessment.
- Double immunodiffusion assays to assess antigenic relationship.
- Enzyme kinetics studies (Km, Vmax).
- N-terminal amino acid analysis and circular dichroism (CD) spectroscopy.
Main Results:
- Purified acid DNase from both young and old brains showed similar molecular weight (62,000) and heat stability.
- Specific activity of old acid DNase was one-tenth that of young acid DNase.
- Old acid DNase exhibited higher Km and lower Vmax.
- CD spectra indicated that old acid DNase molecules are more rigid with increased alpha-helical structure.
- Immunotitration suggested the presence of inactive molecules in the old preparation.
Conclusions:
- The reduced specific activity of old chick brain acid DNase is likely due to conformational changes and increased molecular rigidity.
- These structural alterations may lead to the presence of partially or completely inactive enzyme molecules.
- Age-related modifications in enzyme structure can significantly impact their function.
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 Hg2+, Zn2+, Mg2+, etc., were also similar. The old enzyme showed slightly higher Km and decreased Vmax 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 alpha-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.