Cape baboon Cytochrome P450 11β-hydroxylases : the characterization of two functional enzymes

Brown, Natasja (2007-03)

Dissertation (PhD)--University of Stellenbosch, 2007.

Thesis

ENGLISH ABSTRACT: This study: 1. Describes the localization of CYP11B1 in the Cape baboon adrenal gland using Western blot analysis. CYP11B1 was localized to the adrenal cortex and medulla. 2. Describes the catalytic activity of CYP11B1 towards 11-deoxycorticosterone and corticosterone in adrenal cortical- and medullary tissue homogenates. Aldosterone formation in the adrenal medulla was identified using an atmospheric pressure chemical ionization-mass spectrometry method, which was developed in our department. 3. Compares the catalytic activity of three recombinant Cape baboon CYP11B1 cDNAs, expressed in COS-1 cells, towards 11-deoxycorticosterone and 11-deoxycortisol. 4. Describes the determination of the Michaelis-Menten constants and maximum reaction rates of 11-deoxycorticosterone and 11-deoxycortisol utilization by two functional recombinant Cape baboon CYP11B1 cDNAs, respectively. 11-Deoxycorticosterone metabolites were quantified using an enzyme immunoassay kit. 11-Deoxycortisol metabolites were quantified using a liquid chromatography-mass spectrometry method, which was developed in our department. 5. Describes the homology modeling of two isoforms of Cape baboon CYP11B1 using CYP102 and CYP2C5 as structural templates. The influence of three amino acid residue substitutions, located in the predicted D-E helix, on the catalytic activity of the two CYP11B1 isoforms was examined.

AFRIKAANSE OPSOMMING: Hierdie studie: 1. Beskryf die lokalisering van CYP11B1 in die bynier van die Kaapse bobbejaan deur gebruik te maak van die Western kladtegniek. CYP11B1 is gelokaliseer tot die adrenale korteks en medulla. 2. Beskryf die metabolisme van 11-deoksikortikosteroon en kortikosteroon in adrenale korteks- and medulla weefsel preparate, onderskeidelik. Die produksie van aldosteroon in die medulla is geïdentifiseer deur gebruik te maak van ‘n atmosferiese druk chemiese ionisasie-massa spektrometrie metode wat in ons departement ontwikkel is. 3. Vergelyk die katalitiese aktiwiteit van drie rekombinante Kaapse bobbejaan CYP11B1 cDNAs, getransfekteer in COS-1 selle, ten opsigte van 11-deoksikortikosteroon en 11- deoksikortisol metabolisme. 4. Beskryf die bepaling van die Michaelis-Menten konstantes en maksimum snelhede van twee funksionele rekombinante Kaapse bobbejaan CYP11B1 cDNAs, getransfekteer in COS-1 selle, ten opsigte van 11-deoksikortikosteroon en 11-deoksikortisol metabolisme. 11-Deoksikortikosteroon metaboliete is gekwantifiseer deur gebruik te maak van ‘n ensiem immunotoets. 11-Deoksikortisol metaboliete is gekwantifiseer deur middel van ‘n vloeistofchromatografie-massaspektrometrie metode, ontwikkel in ons departement. 5. Beskryf die modelering van drie-dimensionele strukture van twee funksionele Kaapse bobbejaan CYP11B1 isoensieme deur CYP102 en CYP2C5 as template te gebruik. Die effek van drie aminosuurresiduveranderinge in die voorspelde D-E heliks op die katalitiese aktiwiteit van die twee CYP11B1 isoforme is bepaal.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/19438
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