Δ⁵3β 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)

Characterization of the regenerated Leydig cell population of the rat after destruction by ethylene-1,2-dimethanesulphonate

“…Recent evidence has indicated that 3ß-HSD activity is the second major ratelimiting step (after cholesterol ester cleavage) in the production of testosterone by Leydig cells (Lambert et al 1986). Moreover, kinetic studies on the rat ovarian corpora lutea have revealed that the enzyme exhibits positive co-operative substrate binding, an indication of an enzyme under regulatory control (Gordon & Robertson, 1987). It has been established that steroid¬ ogenic enzymes, including 3ß-HSD, are inducible in steroid-producing cells by the trophic action of pituitary hormones (Ewing & Zirkin, 1983).…”

Rapid continuous monitoring of enzyme activity in tissue sections: Experience with the M85A and Zeiss UMSP-30 systems

“…The use of the M85A microdensitometer for computerized continuous monitoring has been described previously (Robertson et al, 1984;Gordon & Robertson, 1986, 1987.…”

“…In particular methods have been developed for the continuous monitoring of enzyme activity in tissue sections (Kugler, 1981;Robertson et al, 1984). Such techniques allow measurements at a large number of time points to be made as the reaction proceeds and we have described such a method in detail for the monitoring of the activity of ovarian 36-hydroxysteroid dehydrogenase using the Vickers M85A microdensitometer and Commodore PET microcomputer (Robertson et al, 1984;Gordon & Robertson, 1986, 1987.…”