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Δ 5 3β hydroxysteroid dehydrogenase activity of the rat corpus luteum exhibits positive substrate‐binding co‐operativity: A continuous monitoring microdensitometric study with unfixed ovarian tissue sections
Author(s) -
Gordon M.,
Robertson W. R.
Publication year - 1987
Publication title -
cell biochemistry and function
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.933
H-Index - 61
eISSN - 1099-0844
pISSN - 0263-6484
DOI - 10.1002/cbf.290050404
Subject(s) - corpus luteum , dehydroepiandrosterone , dehydrogenase , ovary , endocrinology , medicine , pregnenolone , enzyme , steroid , chemistry , enzyme assay , biology , biochemistry , hormone , androgen
Δ 5 3β hydroxysteroid dehydrogenase activity transforms biologically inactive Δ 5 3β hydroxy steroids into the active Δ 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 Δ 5 3β HSD we have analysed the initial velocity rates ( V o ) for dehydroepiandrosterone (DHEA) binding to this enzyme in regressing (i.e. 20α hydroxy steroid dehydrogenase positive) corpus luteum (CL) cells in unfixed tissue sections (5 μm) of the dioestrous and proestrous rat ovary. The results are mean ± S.E.M. The relationship between DHEA concentration (0 to 50 μM) and Δ 5 3β HSD activity in the dioestrous corpora lutea was sigmoidal and had an atypical 1/ V o versus 1/ S plot, the x intercept being positive. Using a 1/ V o versus 1/ S 2 plot the V max was determined to be 1·0 ± 0·08 μmol 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 μmoles ( n = 6). In the corpora lutea cells of the proestrous ovary, the V max for DHEA transformation was unchanged (0·95 ± 0·04 μmol min −1 mg −1 , n = 3) whilst the S 0·5 was significantly increased to 27 ± 0·1 ( p < 0·01, n = 3). The Hill constant remained positive being 2·9 ± 0·2 ( n = 3). NAD + binding to 3β HSD in regressing corpora lutea of the proestrous ovary has been demonstrated previously to be hyperbolic and fit the classical Michaelis‐Menten model. 1 Extending the analysis of NAD + binding to the regressing corpus luteum of the dioestrous rat ovary revealed similar kinetic characteristics to that seen with the proestrous enzyme, the apparent V max and K m being 0·84 ± 0·04 μmol min −1 mg −1 CL ( n = 3) and 27 ± 7 μmol 1 −1 ( n = 3) respectively. The Hill constant was 1·1 ± 0·03 ( n = 3), indicating no co‐operativity of co‐factor binding.