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

Goosen, Pierre (2012-12)

Thesis (PhD)--Stellenbosch University, 2012.

Thesis

ENGLISH ABSTRACT: This study describes: - the characterization and comparison of the enzymatic activity of both Angora and ovine 3βHSD expressed in non-steroidogenic COS-1 cells. The apparent Km and Vmax values for the metabolism of PREG, 17-OHPREG and DHEA were determined; - the characterization of steroid metabolites produced by COS-1 cells coexpressing either Angora or ovine 3βHSD together with Angora CYP17, in the presence and absence of overexpressed Cyt-b5, following the metabolism of PREG and 17-OHPREG. 3βHSD was identified as an additional 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 coexpressed in non-steroidogenic COS-1 cells; - the influence of purified ovine live Cyt-b5 and anti-Cyt-b5 IgG on adrenal microsomal 3βHSD activity. Cyt-b5 was shown to specifically augment 3βHSD activity which represents the first documentation of such augmentation in any species; - the overexpression and purification of Angora 3βHSD using a baculovirus expression system coupled with a detergent based enzyme purification method; - the characterization of both substrate and co-factor kinetics for the individual dehydrogenase and isomerase activities of purified 3βHSD, in the presence and absence of purified ovine liver Cyt-b5. Cyt-b5 was shown to increase the affinity of 3βHSD towards NAD+ during the dehydrogenase reaction whilst having no significant influence on the isomerase reaction. This represents 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.

AFRIKAANSE OPSOMMING: Hierdie studie beskryf: - die karakterisering en vergelyking van die ensiematiese aktiwiteit van beide Angora en skaap 3βHSD, wat uitgedruk was in nie-steroïed genererende COS-1 selle. Die Km en Vmax waardes tydens die metabolisme van PREG, 17-OHPREG en DHEA was bepaal; - die karakterisering van steroïed metaboliete gegenereer deur COS-1 selle wat Angora of skaap 3βHSD uitdruk saam met Angora CYP17, in die aanwesigheid of afwesigheid van sitochroom b5, na die metaboliseering van PREG en 17-OHPREG. 3βHSD was geïdentifiseer as ‘n bydraende faktor in die oorsaak van hipokortisolisme in die Suid-Afrikaanse Angorabok; - die invloed van sitochroom b5 op die ensiematiese aktiwiteit van beide Angora en skaap 3βHSD wat saam uitgedruk was in nie-steroïed genererende COS-1 selle; - die invloed van gesuiwerde skaap lewer sitochroom b5 en sitochroom b5 teenstof op mikrosomale 3βHSD aktiwiteit. Dit is getoon dat sitochroom b5 die aktiwiteit van 3βHSD spesifiek verhoog. Hierdie studie verteenwoordig die eerste dokumentasie van so ‘n verhoging in enige spesie; - die uitdrukking en suiwering van Angora 3βHSD deur middel van ‘n bakulo-virus sisteem gekoppel aan ‘n detergent gebaseerde ensiem suiwerings metode; - die karakterisering van beide substraat en ko-faktor kinetika vir die afsonderlike dehidrogenase en isomerase aktiwiteite van gesuiwerde 3βHSD, in die aanwesigheid of afwesigheid van gesuiwerde sitochroom b5. Dit is getoon dat sitochroom b5 die affiniteit van 3βHSD teenoor NAD+ tydens die dehidrogenase reaksie verhoog sonder om ‘n beduidende invloed op die isomerase reaksie te hê. Hierdie studie verteenwoordig die eerste dokumentasie van sitochroom b5 wat ko-faktor binding beïnvloed in enige lid van die hidroksisteroïed dehidrogenase familie van ensieme; - die analise van FRET sein in COS-1 selle wat beide 3βHSD-eCFP en Cyt-b5- eYFP fusie proteïene uitdruk. Die resultate stel voor dat sitochroom b5 3βHSD aktiwiteit beïnvloed deur middel van ‘n allosteriese meganisme.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/71775
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