Shogo Ichii

Purification and some properties of cholesterol 20α-hydroxylase from HOG adrenal mitochondria

Abstract 1. 1. A method has been developed for the determination of cholesterol 20αhydroxylase complex using [4- 14 C]cholesterol and 20α-hydroxy[7α- 3 H]cholesterol. 2. 2. An approx. 50-fold purification of the cholesterol 20α-hydroxylase complex from acetone-dried mitochondria of hog adrenal cortex was accomplished by (NH 4 ) 2 SO 4 fractionation, Sephadex G-200 and DEAE-cellulose column chromatography. 3. 3. Addition of non-heme iron-protein as well as NADPH to the incubation mixture was essential for both the 20α- and 22ξ-hydroxylation of cholesterol. 4. 4. The activities of cholesterol 22ξ-hydroxylase and cholesterol C-20-C-22-lyase were not separated from the activity of cholesterol 20α-hydroxylase during the purification of the enzyme. 5. 5. The K m value of the cholesterol 20α-hydroxylase for cholesterol was estimated to be 0.57·10 −5 M at pH 7.4 in the presence of optimum amounts of NADPH and non-heme iron-protein. 6. 6. The influence of pli, metal ions and pregnenolone on the activity of cholesterol 20α-hydroxylase was examined.

Effect of ACTH in vivo on the cholesterol side-chain cleaving enzyme and on steroid hormone precursor cholesterol in rat adrenal gland

Abstract Administration of ACTH in vivo would not appear to stimulate the activity of the cholesterol side-chain cleaving enzyme in rat adrenals. The apparent increased activity of the enzyme observed following the administration of ACTH should be attributed to the decreased amount of “endogenous precursor cholesterol”. The radioactivity of the cholesterol added to the incubation mixture was found to be concentrated in the large particulate fraction of rat adrenal homogenate after the incubation and an attempt to assess the amount of “endogenous precursor cholesterol” was described.

Studies on the side-chain cleavage of C21-steroids

Incubation studies of hog adrenal and rat testis homogenates with 3H-17α-hydroxyprogesterone and 14C-pregn-4-ene-17α, 20α-diol as a mixture revealed differences in pathways between these two tissues in the formation of C19-steroids from the C21-precursors. The 3H14C ratios found in the isolated pregn-4-ene-17α, 20α-diol-3-one, 17α-hydroxyprogesterone, androst-4-ene-3,17-dione and testosterone suggested that in the hog adrenal homogenate the C19-steroids were derived mainly from pregn-4-ene-17α, 20α-diol-3-one while 17α-hydroxyprogesterone seemed to be the immediate precursor of the C19-steroids in the rat testis homogenate.

In vitro effect of corticotropin on the activity of the phosphorylase of rat adrenal

Abstract It has been suggested that ACTH stimulates phosphorylase of adrenal cortex. The present paper tests the possibility of in vitro stimulation of phosphorylase by ACTH with adrenals from intact rats. Bisected adrenals were incubated with or without ACTH. ACTH prevented the loss of phosphorylase activity when added to the incubation medium without preincubation or after short-term preincubation, but did not show any effect on phosphorylase when added after longer preincubation time. However, it was evident that ACTH stimulates adrenal steroid synthesis even when added after 1 hour preincubation. These results indicate that phosphorylase is not obligatory for the increased production of corticosteroids under the influence of ACTH in rat adrenals in vitro .

In vivo effects of corticotropin on the effective concentration of phosphorylase and glucose-6-phosphate dehydrogenase in rat adrenal

Abstract The effective concentration of phosphorylase and glucose-6-phosphate dehydrogenase in rat adrenals has been determined after the injection of ACTH. Large doses of ACTH, 16 and 28 I.U./kg. body weight, did not affect the levels of either enzyme. A massive dose,56 I.U./kg. body weight, resulted in a remarkable fall in phosphorylase and a slight decrease in glucose-6-phosphate dehydrogenase. These results indicate that ACTH does not necessarily exert its stimulatory effect on rat adrenal in vivo through stimulation of phosphorylase and hexose monophosphate shunt at the level of glucose-6-phosphate dehydrogenase.