Richard B. Young

Studies of the oxidation state of partially purified adrenal cortex mitochondrial cytochrome P-450 and difference spectra induced by deoxycorticosterone and metopirone.

Abstract Cytochrome P-450 prepared from extracts of adrenal gland cortex mitochondrial acetone powder exists in the oxidized state as it will not complex with carbon monoxide. Both deoxycorticosterone and Metopirone will induce spectra in the preparation in the absence of oxygen and reductase components. Metopirone displaces the deoxycorticosterone-induced difference spectrum of the cytochrome P-450 preparation, but excess deoxycorticosterone does not influence the Metopirone-induced spectrum suggesting different complexing mechanisms for the two compounds. The observations are consistent with the concept that the steroid-induced spectrum is the result of a direct complexing with ferric cytochrome P-450, or a closely associated companion pigment dependent upon the ferric state of cytochrome P-450. Current observations indicate experimentally separable reaction steps in the overall system of 11β-hydroxylation.

Preparation of a soluble progesterone 17α-hydroxylating system

Abstract Examination of differentially centrifuged homogenates of bovine adrenocortical tissue reveals that the steroid 17α-hydroxylating system resides primarily in the particle-free cytoplasm. Preparations are relatively labile. Higher activity is noted in fresh unfrozen glands than from frozen ones. 17-Hydroxylation is inhibited less in higher pH media and in media containing p -chloromercuribenzoate than is 21-hydroxylation.

Steroid 17α-hydroxylation in the rat adrenal gland

Abstract A 17α-hydroxylating system has been demonstrated conclusively for the first time in normal rat adrenal tissue. The system is precipitated from extracts of adrenal tissue at 40% saturation of ammonium sulfate. When this fraction is incubated with protein components precipitated at 80% saturation of ammonium sulfate, only 11β-hydroxylation occurs. It is concluded that the normal rat adrenal gland has the capacity to 17α-hydroxylate progesterone, but that this reaction as studied in vitro is inhibited in the presence of the 11β-hydroxylating system. To what extent this inhibition occurs in situ is not known.

Low magnitude transformation of estradiol to estrone in human endometrium

Abstract 1. 1. The human endometrium estradiol dehydrogenase has been shown to function at magnitudes significantly lower than those in which estradiol binding has been examined. Quantities as low as 0.0075 fmole have been shown to be transformed to 0.006 fmole of estione in 1 min by 1 mg of tissue. 2. 2. In sonicated preparations, transformation of estradiol to estrone is enhanced by NAD + and NADP + . NADPH does not influence the reverse reaction. 3. 3. The dehydrogenase appears to be cold sensitive, conforming to the observations of Jarabak et al. (Jarabak, J., Seeds, A. S., Jr and Talahay, P., (1966) Biochemistry 5, 1269) with the placental estrogen dehydrogenase.

Two adrenal gland cytochrome P-450 entities evidenced by metyrapone-induced spectra

Abstract 1. Marked diminution in the metyrapone-induced 425-nm difference spectrum occurs when 17-hydroxyprogesterone is added to microsomal preparations, but only a low magnitude response is observed when this steroid is introduced into mitochondrial fractions exhibiting metyrapone spectra. Deoxycorticosterone induces spectra in both acellular fractions, but does not influence the magnitude of metyrapone spectrum. 2. Diminution of the microsomal metyrapone absorption peak after addition of 17-hydroxyprogesterone, appears to be primarily due to summation of the 420-nm absorption minimum induced by 17-hydroxyprogesterone and the 425-nm absorption maximum induced with metyrapone. 3. Metyrapone cancels deoxycorticosterone-induced spectra occurring either in microsomes or mitochondria and attains a magnitude identical to that attained in the absence of deoxycorticosterone. 4. Addition of metyrapone to microsomal preparations containing deoxycorticosterone results in the same difference spectrum as that manifest by metyrapone alone, suggesting complete displacement of the spectral inducing elements of deoxycorticosterone in preference to those of metyrapone. Such a manifestation, however, does not occur in respect to 17-hydroxyprogesterone. The spectral shift which occurs when metyrapone is added to preparations containing 17-hydroxyprogesterone is distinct from metyrapone alone. When metyrapone is added to the reference cuvette, the sample cuvette reveals the 17-hydroxyprogesterone spectrum; when 17-hydroxyprogesterone is added to the reference cuvette the metyrapone spectrum is revealed indicating independence of the two substrates in inducing their respective difference spectra.

Biosynthesis of steroid hormones in a human feminizing adrenal carcinoma.

Pregnenolone‐4‐14C, progesterone‐4‐14C, 17α‐hydroxyprogesterone‐4‐14C, 11‐deox‐ycorticosterone‐4‐14C, androstenedione‐4‐14C and testosterone‐4‐14C were incubated with a homogenate of a feminizing human adrenocortical carcinoma. Estrone, 17β‐estradiol were isolated and identified in the phenolic fraction from substrates: pregnenolone, progresterone, androstenedione and testosterone. From androstenedione 19‐hydroxyandrostenedione and 19‐nortestosterone were identified as metabolic products. Hydroxylation reactions were active for carbons 6, 17 and 21 and appeared to proceed at rates normal to adrenal gland tissue; 16‐hydroxylation was not observed. In the tissue homogenates 11β‐hydroxylation was not observed; however, it could be demonstrated when supported with the addition of components from bovine adrenocortical mitochondria, thus indicating the presence of only part of the 11β‐hydroxylating system.

II-deoxysteroid-induced spectral responses in adrenal gland microsomes

Abstract 1. 1. Evidence derived both from spectrometric and incubation studies shows that microsomes prepared by contemporary methods of differential centrifugation exhibit appreciable mitochondrial-like activity. This is particularly demonstrable in incubation studies where the addition of the mitochondrial cytochrome P-450 reductase system (which is soluble and is extracted from the system by conventional produres) markedly increases II β-hydroxylation. The presence of significant quantities of mitochondrial-like cytochrome P-450 in microsomal preparations offers explanations for spectral responses to various steriod substrates observed in microsomal preparations. Induced difference spectra occur in these preparations both with II -deoxysteroid and 21-deoxysteroid substrates. 2. 2. The magnitude of spectral response induced by androstenedione in microsomal preparations is markedly different in comparison to that induced by II -deoxycorticosterone. Androstenedione induces the greatest absorption minimum at 420 nm. After inducing the maximal spectral response possible with II -deoxycorticosterone, subsequent addition of androstenedione results in a deepening of the absorption minimum at 420 nm. This suggests that androstenedione, which serves as a substrate for II β-hydroxylation, responds with both mitochondrial and microsomal cytochrome P-450 moieties in these preparations. The former seems likely associated with the II β-hydroxylation reaction, the latter remains unexplained.