Inhibition by 2'-deoxyadenosine of enzyme induction in Pseudomonas testosteroni.

Among various systems in which induction of new enzyme synthesis can be initiated, that developed by Talalay,'-3 in which the metabolism of a variety of steroids by Pseudomonas testosieroni is induced by either testosterone or certain other steroidal compounds, has been particularly rewarding. Recent studies of this system by Wacker et al.4' 5 have suggested that a complex may be formed between the inducer, in this case testosterone, and repressors, possibly proteins,4 thus leading to derepression and the formation of the appropriate ribonucleic acids and specific enzyme proteins. Although testosterone is metabolized rapidly and completely by Pseudomonas testosteroni, the inducible enzymes so far extensively studied catalyze only early steps in catabolism; these enzymes are 3#, 17,3-hydroxysteroid dehydrogenase, 3a-hydroxysteroid dehydrogenase, and A5-3-ketosteroid isomerase.1-5 Certain cytotoxic substances, e.g., 5-fluorouracil, mitomycin C, puromycin, pfluorophenylalanine, and ethionine, appear to affect the induced formation of enzymes more than processes involved in the reproduction of the microorganism;5 however, no reports of the effects of either natural or unnatural nucleosides on the induction process have appeared. This paper is concerned with studies of the inhibition of induction of certain enzyme activities, particularly z5-3-ketosteroid isomerase, in Pseudomonas testosteroni, caused by the addition to the medium of 2'deoxyadenosine, an effect much more marked than that observed with other nucleosides or bases tested, including 3'-deoxyadenosine and adenosine. Materials and Methods.-The procedures used differed but little from those employed by Marcus and Talalay2 I and by Wacker et al.4 I Pseudonwnas testosteroni (ATCC 11996; the majority of the experiments were carried out with a culture kindly provided by Prof. P. Talalay) was grown in a medium of pH 7 with a composition similar to that described by Wacker et al.,5 except that sodium lactate (5 gm per liter) was employed instead of glucose, and casamino acids and yeast extract (both obtained from the Difco Laboratories) were present at the level of 5 gm of each per liter. The organisms used as an inoculum were taken from an agar slant and grown overnight at 300 in a liquid medium (without steroid or amino acids); of this culture, 4.3 ml were added to each 100 ml of growth medium at the beginning of each experiment. Incubation of 5.0 ml of the inoculated medium was carried out for 7 hr in conical fermentation flasks (125 ml) at 30° in a gyrorotatory shaker (New Brunswick Scientific Co.) operated at 170 4 20 oscillations per min. In all cases, the inducer, M-androstene-3,17-dione, 1.25 mg dissolved in dimethylsulfoxide (DMS0), 0.05 ml, was added 2 hr after the initiation of incubation. In concentrations up to at least 5%, DMSO had no detectable effect on either the growth of the organism or the induction of enzyme activities. Adenine sulfate (Nutritional Biochemicals Corp. #4829), adenosine (Pabst #1408), and various lots of 2'-deoxyadenosine or its monohydrate (the most satisfactory material was that of Calbiochem, #590893) were added to the flasks either as solids or as solutions in DMS0, of which not more than 0.1 ml was necessary per 5 ml of culture medium. 3'-Deoxyadenosine (generously provided by Dr. H. Klenow), di-sodium 2'-deoxyadenylate (5') (Calbiochem #30475), and the other 2'-deoxyribonucleosides tested (obtained from various sources) were added at appropriate times to the culture flasks as solid substances. After removal of the flasks from the incubator, the turbidity of dilutions of the cultures was