Determination of cytochrome P-450 in Cunninghamella elegans intact protoplasts and cell-free preparations capable of steroid hydroxylation
✍ Scribed by Jerzy Dlugoński; Katarzyna Bartnicka; Iwona Zemelko; Violetta Chojecka; Prof. Dr. Leon Sedlaczek
- Book ID
- 102910361
- Publisher
- John Wiley and Sons
- Year
- 1991
- Tongue
- English
- Weight
- 639 KB
- Volume
- 31
- Category
- Article
- ISSN
- 0233-111X
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✦ Synopsis
Cytochrome P-450 was shown to be involved in 1 la-, and lib-hydroxylation of Substance S in intact C. elegans protoplasts. The steroid transformation was inhibited by carbon monoxide, the inhibitory effect being dependent on CO concentration. The function of cyt P-450 in intact protoplasts was confirmed by the estimation of strong absorption at 450 nm in the CO difference spectrum. The presencc of antimycin A was necessary to prevent the reduction of the cytochrome oxidase and its interference with the cyt P-450 in the spectrophotometric analysis.
The intracellular content of cyt P-450 could be increased from 5.25 pM/mg protein to 26.88 pM/mg protein when the steroid inducer was present in the medium at each stage of protoplast preparation and during cyt P-450 determination.
The enriched microsomal fraction obtained from the crude extract of ruptured protoplasts contained the steroid 1 la-hydroxylase system of C. elegans. The activity of 1 lp-hydroxylase could not be detected under the conditions of the experiment. The localization of steroid hydroxylases of C. efeguns in microsomes was confirmed by cyt P-450 detection in the 9600 x g supernatant. Membranous fractions (pellets 1100 x g and 9600 x g) of the concanavaline A stabilized protoplasts, carrying the marker plasma-membrane-bound and mitochondria1 ATPases, did not show maximum absorption at 450 nm in the CO difference spectrum.