Androgen Biosynthesis in the Testis
THESIS ABSTRACT The kinetic parameters of testicular steroid synthesizing enzymes involved in the 6 4 and 6 5 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, (k 0 _ 5 >, and Vmax values were determined for 3 B-Hydroxysteroid oxidoreductase (EC 220.127.116.11), 17
... 145), 17 a-Hydroxylase (EC 18.104.22.168) and c 17 -c 20 Lyase reactions using 6 4 and 6 5 substrates. The purpose of the study was to develop a kinetic model for predicting the predominant steroidogenic pathway in roammalian testes. The key step in determining the direction of the pathway is the conversion of pregnenolone to either progesterone (6 4 ) or to 17a -hydroxypregnenolone (6 5 ). The most definitive kinetic predictors were found to be 1) the Michaelis constant of the 3BHSOR reaction for PREG and, 2) the V of max the PREG l? a -Hydroxy lase reaction. The 6 4 species demonstrate a low k 0 _ 5 for PREG in the 3BHSOR reaction and a low Vmax for the conversion of PREG-tl?a PREG. Conversely, 6 5 species demonstrate a high k 0 . 5 for PREG in the 3BHSOR reaction and a high V for the hydroxylation. The relative affinity max of 3BHSOR and l?a -Hydroxylase for PREG was found to be a useful predictor of the predominant p athway . A high ratio, indicating greater affinity of the hydroxylase for PREG, favors conversion to 6 5 steroids and a low ratio is indicative of the 6 4 pathway. Several species were shown to employ mixed 6 4 and 6 5 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 lyaseactivities as four distinct proteins. Additionally, a soluble c 17 -c 20 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 varia.is 6 5 substrates by the hypovitaminosis C pretreatment. Partial reversal of inhibition could be obtained by adding ascorbate or dehydroascorbate to in vitro incubations.