Characterization of the Basis for Differences in Serum DBH Activity in Immature and Adult Rats by Use of Homologous Antibody

Abstract

Rat serum dopamine-8-hydroxylase (DBH) activity decreased 5-7-fold between 15 and 60 days of age. Immunoprecipitation performed with homologous antibody (guinea-pig anti-rat adrenal DBH) showed that during this time period the quantity of antibody necessary to precipitate 50% of the enzymatic activity (AD,,) decreased 5-fold from 0.25 to 0.05 pl/ml. The biochemical properties of rat serum DBH at 15 and 60 days of age were compared to test the hypothesis that there might be different biochemical forms of the enzyme in the blood of immature and adult rats. Thermal stability, apparent K, for tyramine, electrophoretic mobility, pH optima and elution profile on gel filtratioh chromatography were all found to be similar for rat serum DBH at both ages. On the basis of homospecific activity and multiple similarities in biochemical characteristics, it appears that differences in serum activity at the two ages reflect differences in the steady-state levels of enzyme.To determine the turnover of serum DBH in the two age groups, the recovery of enzyme activity was monitored after acute clearance of the circulating pool of DBH by treatment with the homologous antiserum. Immuno-titration of DBH activity in uiuo indicated that the total pool of serum enzyme was 4-fold greater in the mature rat than in 4-day-olds. After treatment of adult rats with 2pl of homologous antiserum, serum DBH activity was reduced by 85% with a half-life of recovery of 3.0 f 0.6 days; the estimated fractional rate of degradation was 0.23 f 0.06day-' and the rate of entrance was 2.3 f 0.2 units/ml/'day. After treatment of 4-day-old rats with 1 p1 of homologous antiserum, serum DBH activity was reduced by 95% with a half-life of recovery of 3.3 + 0.5 days: the estimated average fractional rate of degradation was 0.22 0.06day-' and the average rate of entrance was 10.7 & 1.6 units/ml/day. Thus, the several-fold difference in steady-state levels of serum DBH in rat pups as compared to adult rats appears to be due to greatly increased rates of entrance of the enzyme in the immature rats.