S.L. Hsia

Photoreactivation spectrum of the co-inhibited taurochenodeoxycholate 6β-hydroxylase system☆

The 6&hydroxylation of chenodeoxycholic and taurochenodeoxycholic acid by cell free preparations of rat liver has been reported in a previous paper [l] . The enzymic activity was located in the microsomal fraction, and an active extract was prepared by suspension of the microsomes in 1 .O M phosphate buffer, pH 7.6 and subsequent centrifugation at 105,000 X g. The requirements for NADPH and 0, indicated that the 6@hydroxylase is a mixed function oxidase [Z] . Carbon monoxide was found to inhibit the hydroxylase activity of the 1 .O M phosphate extract and the inhibition could be partially reversed by white light. This, along with the spectrophotometric detection of cytochrome P-450 in the extract [I] , following reduction with Na dithionite and exposure to CO, suggested the participation of the P-450 in the 6&hydroxylase system. In a continuation of our study we have determined the photoreactivation spectrum of the COinhibited hydroxylase system. This communication reports the results which support that P450 functions as the oxygen activating enzyme in the G/3-hydroxylase system.

Lipid Synthesis from Acetate-1-14C by Suction Blister Epidermis and Other Skin Components

Summary Lipids synthesized from acetate-1-14C in vitro in human dermis, epidermis, and sebaceous glands were examined. On the average, epidermis incorporates more 14C into lipids than dermis does in a given specimen. On per milligram basis, epidermis is several times more active than dermis. The lipids were separated into eight classes by chromatography on Unisil and Florisil columns. 14C was found in all lipid classes. The major amounts of 14C were found in the sterols, glycerides, and polar lipids of both dermis and epidermis, but greater proportions of 14C were found in the sterol and polar lipid fractions of epidermis than in corresponding fractions of dermis. Synthesis of squalene and triglycerides appeared to characterize the activity of the sebaceous glands. The epidermis excised from suction blisters was found active in synthesizing lipids from acetate-l-14C, and the activity appeared to increase after the ingestion of 100 g of glucose, indicating metabolic regulation of lipid synthesis in the epidermis.

Transformation of dehydroepiandrosterone into dihydrotestosterone by isolated cells from rat preputial gland.

After the rat preputial gland was treated with collagenase and trypsin, five bands of cells were isolated by centrifugation in Ficoll gradients. Homogenates of the heavier cells (Bands IV and V) which contained less lipids, were more active than the homogenates of the lighter cells (Bands I, II and III) in transforming [1,2-3H]-dehydroepiandrosterone ([1,2-3H]-DHA) into [3H]-androstenedione and [3H]-testosterone and the latter into [3H]-dihydrotestosterone (DHT). In the presence of NAD, NADH and NADPH-generating system, [1,2-3H]-DHA was transformed into [3H]-DHT in 50-60% yield by homogenates of cells in Bands IV and V. DHT levels in the preputial gland were measured by radioimmunoassay. The levels in female rats reduced by 77% from 3.14 +/- 0.27 to 0.72 +/- 0.10 pg/mg tissue after adrenalectomy, and by 45% to 1.71 +/- 0.10 pg/mg tissue after ovariectomy. In male rats, the level reduced by 15% from 4.58 +/- 0.55 to 3.88 +/- 0.62 pg/mg tissue after adrenalectomy and by 40% to 2.74 +/- 0.21 pg/mg tissue after orchidectomy. These results demonstrated the transformation of DHA into DHT in the preputial gland of the rat, and that the adrenal is an important source of precursor steroid (DHA) for DHT formation in the preputial gland.

P-450 Level and Taurochenodeoxycholate 6β-Hydroxylase System of Rat Liver Microsomes

Summary The level of cytochrome P-450 and the activity of taurochenodeoxycholate 6β-hydroxylase of rat liver microsomes were determined after the administration of phenobarbital, cholestyramine, thyroxine and chenodeoxycholate. Treatment with phenobarbital caused increases in both the 6β-hydroxylase activity and P-450 level, while the other compounds caused decreases in both. Quantitatively there was a lack of parallelism between the hydroxylase activity and the level of P-450, indicating that P-450 is not a rate-limiting factor in the 6β-hydroxylase system. An extract of the microsomes was prepared in 1.0 M phosphate (pH 7.6) as previously described. The extract was active in 6β-hydroxylation, with an apparent Michaelis-Menten constant towards taurochenodeoxycholate of 1.2 × 10-4M, and a maximum rate of hydroxylation of approximately 1 nmole/min/mg protein. The ratio of P-450 to protein of the extract was 2 to 2.5 times greater than that in the microsomes.