The influence of 3βHSD on adrenal steroidogenesis and the factors which influence its activity

[摘要] ENGLISH ABSTRACT: This study describes:- the characterization and comparison of the enzymatic activity of both Angora and ovine 3βHSDexpressed in non-steroidogenic COS-1 cells. The apparent Km and Vmax values for themetabolism of PREG, 17-OHPREG and DHEA were determined;- the characterization of steroid metabolites produced by COS-1 cells coexpressing either Angoraor ovine 3βHSD together with Angora CYP17, in the presence and absence of overexpressedCyt-b5, following the metabolism of PREG and 17-OHPREG. 3βHSD was identified as anadditional factor in causing hypocortisolism in the South African Angora goat;- the influence of Cyt-b5 on the enzymatic activity of both Angora and ovine 3βHSD coexpressedin non-steroidogenic COS-1 cells;- the influence of purified ovine live Cyt-b5 and anti-Cyt-b5 IgG on adrenal microsomal 3βHSDactivity. Cyt-b5 was shown to specifically augment 3βHSD activity which represents the firstdocumentation of such augmentation in any species;- the overexpression and purification of Angora 3βHSD using a baculovirus expression systemcoupled with a detergent based enzyme purification method;- the characterization of both substrate and co-factor kinetics for the individual dehydrogenaseand isomerase activities of purified 3βHSD, in the presence and absence of purified ovine liverCyt-b5. Cyt-b5 was shown to increase the affinity of 3βHSD towards NAD+ during thedehydrogenase reaction whilst having no significant influence on the isomerase reaction. Thisrepresents the first documentation of Cyt-b5 influencing co-factor binding in any member of the-ydroxysteroid dehydrogenases;- the FRET analysis of COS-1 cells coexpressing 3βHSD-eCFP and Cyt-b5-eYFP fusion proteins,suggesting an allosteric interaction between 3βHSD and Cyt-b5.

[发布机构] Stellenbosch University