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.

Protoplasts ofCunninghamella elegans, showing 11α-, and 11β-hydroxylating ability of Substance S, preserved high transformation activity when dispersed in glucose-enriched, organic osmotic stabilizers. A joint action of polyoxins and 2-deoxy-D-glucose was necessary to prevent regeneration of the cell wall in long-lasting experiments. Stabilized and active, dispersed protoplasts may be an alternative research model for studying the function of the cell wall and intracellular metabolic pool constituents in steroid hydroxylation.