The high speed supernatant of mouse liver is active in the C-20 ketone reduction of a number of various steroids (l).
The 20α-hydroxysteroid dehydrogenase responsible for the C-20 ketone reduction of corticosterone and 11-dehydrocorticosterone could be purified by calcium phosphate gel and ammonium sulphate fractionation. The activity was absorbed on the gel between the ratios 2:1 and 6:1 (gel/enzyme preparation - w/w) and eluted with .05 M phosphate buffer resulting in a recovery of 49.6% of the original activity. This preparation was then further purified with ammonium sulphate, the enzyme activity being recovered in the 48 -70% fraction. The overall recovery of corticosterone 20α-hydroxy-steroid dehydrogenase was 34.1% with a 5. fold purification.
Acetone or acid fractionation and Sephadex G-75 filtration did not lead to further purification.
The activity could also be recovered from the crude preparation by fractionation on a Sephadex G-100 column. 87% of the original activity was recovered in one pooled fraction giving a purification of 3.3 fold.
The 20a-hydroxysteroid dehydrogenase reducing 11β-hydroxy-progesterone could be separated from the corticosterone 20α-hydroxy-steroid by calcium phosphate gel fractionation. The former activity was not eluted by 0.05 M phosphate buffer but came off in phosphate buffers of higher ionic strength. No definite conclusions as to separation on Sephadex G-100 columns were obtained due to the in-activation of the 11β-hydroxyprogesterone 20α-hydroxysteroid
dehydrogenase, which could be partly reduced by the presence of NADPH₂. This enzyme was somewhat unstable, being inactivated by 3 day dialysis against running tap water and filtration on a Sephadex G-25 column while the corticosterone 20α-hydroxysteroid dehydro genase was little affected by these procedures.
The multiple nature of the 20α-hydroxysteroid dehydrogenases was also shown by a comparison of the ratio of products from various enzyme fractions. The ratio of 20α-dihydro-11-dehydro corticosterone to 20α-dihydrocorticosterone after incubations with 11-dehydrocorticosterone and corticosterone respectively remained constant indicating one enzyme. However, the ratio of 20α-dihydro-11-dehydrocorticosterone to 20α-dihydro-11β-hydroxyprogesterone after incubations with 11-dehydrocorticosterone and 11β-hydroxy-progesterone respectively varied greatly indicating that these two substrates were reduced by different 20α-hydroxysteroid dehydrogenases. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate
| Identifer | oai:union.ndltd.org:UBC/oai:circle.library.ubc.ca:2429/36816 |
| Date | January 1966 |
| Creators | Deeth, Leslie A.M. |
| Publisher | University of British Columbia |
| Source Sets | University of British Columbia |
| Language | English |
| Detected Language | English |
| Type | Text, Thesis/Dissertation |
| Rights | For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use. |
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