Date of Award

1982

Degree Type

Dissertation

Specialization

Food Science and Nutrition

Department

Food Science and Nutrition

First Advisor

James G. Bergman

Abstract

The kinetic parameters of testicular steroid synthesizing enzymes involved in the Δ4 and Δ5 pathway s for testosterone production were examined in four species. Testis microsomes were prepared as a source of steroidogenic enzymes. Radiolabeled steroid-intermediates were used as substrates and products were separated by thin layer chromatography. Michaelis constants, (k0.5), and Vmax values were determined for 3 B-Hydroxysteroid oxidoreductase (EC 1.1.1.145), 17α-Hydroxylase (EC 1.14.99.9) and C17-C20 Lyase reactions using Δ4 and Δ5 substrates.

The purpose of the study was to develop a kinetic model for predicting the predominant steroidogenic pathway in mammalian testes. The key step in determining the direction of the pathway is the conversion of pregnenolone to either progesterone (Δ4) or to 17α-hydroxypregnenolone (Δ5). The most definitive kinetic predictors were found to be 1) the Michaelis constant of the 3BHSOR reaction for PREG and, 2) the Vmax of the PREG l7α-Hydroxy lase reaction. The Δ4 species demonstrate a low k0.5 for PREG in the 3BHSOR reaction and a low Vmax for the conversion of PREG -> 17αPREG. Conversely, Δ5 species demonstrate a high k0.5 for PREG in the 3BHSOR reaction and a high V for the hydroxylation. The relative affinity max of 3BHSOR and l7α-Hydroxylase for PREG was found to be a useful predictor of the predominant pathway. A high ratio, indicating greater affinity of the hydroxylase for PREG, favors conversion to Δ5 steroids and a low ratio is indicative of the Δ4 pathway. Several species were shown to employ mixed Δ4 and Δ5 pathways in the production of testosterone.

Differing kinetic parameters and the absence of lyase activity in the opposite, (nonfavored), pathway in the dog, rabbit and guinea pig are evaluated as evidence for the existence of hydroxylase and lyase activities as four distinct proteins. Additionally, a soluble C17-C20 lyase, with dual nucleotide specificity, was identified in the dog. Regulation of testosterone biosynthesis is discussed in relation to hydroxylase and lyase activity in the four species.

Similar procedures were employed to evaluate the role of ascorbic acid in steroid synthesis. Guinea pigs with latent hypovitaminosis C were used as a model for evaluation of the effect of ascorbic acid on 3 BHSOR activity. The enzyme was inhibited 37-76% for the various Δ5 substrates by the hypovitaminosis C pretreatment. Partial reversal of inhibition could be obtained by adding ascorbate or dehydroascorbate to in vitro incubations.

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