Determination of cytochrome P-450 in Cunninghamella elegans intact protoplasts and cell-free preparations capable of steroid hydroxylation.
Cytochrome P-450 was shown to be involved in 11 alpha-, and 11 beta-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 presence 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 11 alpha-hydroxylase system of C. elegans. The activity of 11 beta-hydroxylase could not be detected under the conditions of the experiment. The localization of steroid hydroxylases of C. elegans 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 mitochondrial ATPases, did not show maximum absorption at 450 nm in the CO difference spectrum.
Dlugoński
J
J
Microbiology and Virology Centre, Polish Academy of Sciences, Lódź.
Bartnicka
K
K
Zemełko
I
I
Chojecka
V
V
Sedlaczek
L
L
D000968:Antimycin A / Q000494:pharmacology; D002474:Cell-Free System; D008411:Chytridiomycota / Q000378:metabolism*; D003208:Concanavalin A / Q000494:pharmacology; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis; D015971:Gene Expression Regulation, Enzymologic; D010879:Piperazines / Q000378:metabolism*; D011523:Protoplasts / Q000187:drug effects / Q000378:metabolism; D013053:Spectrophotometry; D013252:Steroid 11-beta-Hydroxylase / Q000032:analysis*; D013250:Steroid Hydroxylases / Q000032:analysis; D013347:Subcellular Fractions / Q000201:enzymology
D010879:Piperazines; D003208:Concanavalin A; C012600:substance S; D000968:Antimycin A; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D013252:Steroid 11-beta-Hydroxylase; C074514:steroid 11-alpha-hydroxylase