Delta (5)3 beta hydroxysteroid dehydrogenase activity of the rat corpus luteum exhibits positive substrate-binding co-operativity: a continuous monitoring microdensitometric study with unfixed ovarian tissue sections.

Abstract

delta (5)3 beta hydroxysteroid dehydrogenase activity transforms biologically inactive delta (5)3 beta hydroxy steroids into the active delta (4)3-keto products (e.g. pregnenolone to progesterone). Using a cytochemical procedure which allows for the continuous microdensitometric monitoring of an enzyme reaction as it proceeds and a well described cytochemical assay for delta (5)3 beta HSD we have analysed the initial velocity rates (Vo) for dehydroepiandrosterone (DHEA) binding to this enzyme in regressing (i.e. 20 alpha hydroxy steroid dehydrogenase positive) corpus luteum (CL) cells in unfixed tissue sections (5 micron) of the dioestrous and proestrous rat ovary. The results are mean +/- S.E.M. The relationship between DHEA concentration (0 to 50 microM) and delta (5)3 beta HSD activity in the dioestrous corpora lutea was sigmoidal and had an atypical 1/Vo versus 1/S plot, the x intercept being positive. Using a 1/Vo versus 1/S2 plot the Vmax was determined to be 1.0 +/- 0.08 mumol min-1 mg-1 CL (n = 6). The Hill constant was 2.7 +/- 0.02 (n = 6) suggesting a high degree of positive co-operativity for DHEA binding. The S concentration for half maximal activity was 17 +/- 1 mumoles (n = 6). In the corpora lutea cells of the proestrous ovary, the Vmax for DHEA transformation was unchanged (0.95 +/- 0.04 mumol min-1 mg-1, n = 3) whilst the S0.5 was significantly increased to 27 +/- 0.1 (p less than 0.01, n = 3). The Hill constant remained positive being 2.9 +/- 0.2 (n = 3).(ABSTRACT TRUNCATED AT 250 WORDS)