No abstract

Non‐transformed steroid receptors have an approximately 8S sedimentation coefficient that corresponds to an oligomeric structure of 250‐300 kd which includes a non‐hormone binding 90‐kd protein. A monoclonal antibody BF4 raised against the purified, molybdate‐stabilized, 8S progesterone receptor (8S‐PR) from chick oviduct, recognizes 8S forms of all steroid hormone receptors. BF4 was found specific for a 90‐kd protein present in great abundance in all chicken tissues, including that present in 8S‐forms of steroid receptors. Here, using immunological and biochemical techniques, we demonstrate that this ubiquitous BF4‐positive 90‐kd protein is in fact the chicken 90 kd heat‐shock protein (hsp 90): it increased in heat‐shocked chick embryo fibroblasts, and displayed identical migration in two‐dimensional gel electrophoresis and the same V8 peptide map as the already described hsp 90. We discuss the possibility that the interaction between hsp 90 and steroid hormone‐binding subunits may play a role in keeping the receptor in an inactive form.

Oligodendrocyte mitochondria from 21-dayold Sprague-Dawley male rats were incubated with 100 nM[3lH]cholesterol. It yielded [3lH]pregnenolone at a rate of 2.5 ± 0.7 and 5-[3Hjpregnene-3fi,20a-diol at a rate of 2.5 ± 1.1 pmol per mg of protein per hr. Cultures of glial cells from 19-to 21-day-old fetuses (a mixed population of astrocytes and oligodendrocytes) were incubated for 24 hr with [3H]mevalonolactone.[3H]Cholesterol, [3H]pregnenolone, and 5-[3HJpreg-nene-3fi,20a-diol were characterized in cellular extracts. The formation of the 3H-labeled steroids was increased by dibutyryl cAMP (0.2 mM) added to the culture medium. The active cholesterol side-chain cleavage mechanism, recently suggested immunohistochemically and already observed in cultures of C6 glioma cells, reinforces the concept of "neurosteroids" applied to A5-3fi-hydroxysteroids previously isolated from brain.We have characterized two A5-3f3-hydroxysteroid metabolites of cholesterol, pregnenolone (3,8-hydroxy-5-pregnene-20-one) and dehydroepiandrosterone (3f3-hydroxy-5-androstene-17-one), in the brain of several mammalian species (rat, mouse, monkey, and occasionally pig and human). Definitive identification of the steroid moiety was made in rat brain extracts by gas/liquid chromatography-mass spectrometry (1-3). Pregnenolone has also been identified in acetone powder of rabbit brain by its mass spectrum (4). The A5-3p8-hydroxysteroids persisted in brain after removal of steroidogenic organs, and we therefore proposed that their formation or accumulation in the rat brain depends on in situ mechanisms unrelated to the peripheral endocrine gland system. However, both S. Lieberman's and our group were unable up to now to conclusively demonstrate the biosynthesis of pregnenolone in brain (3, 4). Pregnenolone is the key steroid synthesized from cholesterol in steroidogenic glands (5, 6). The oxidative side-chain cleavage of cholesterol is operated by a specific enzyme complex, which includes cytochrome P-450,CC (7). We have used specific antibodies to the bovine adrenal P-450 for the immunohistochemical localization of the enzyme in the adult rat brain, and we have found that the white matter was selectively immunostained throughout the brain (8 . The latter is a competitive inhibitor of 3,3-hydroxysteroid dehydrogenase activity, which might decrease pregnenolone levels in our experiments. The final protein concentration was 0.8 mg/ml for brain (corresponding to -30 x 106 cells) and 1-2 mg/ml for adrenal mitochondria.[3H]Cholesterol was added (5 x 106 cpm, 0.1 nmol) after suspension in 0.1 ml of Tris buffer (pH 7.4) containing 20 ,ug of Tween 80 (13). The reaction was initiated by the addition of 10 mM isocitrate to generate NADPH. Alternatively, a NADPHgenerating system was used: 0.75 mg of NADP+, 2.5 mg of glucose 6-phosphate, 0.5 unit of glucose-6-phosphate dehydrogenase (from Bakers' yeast; 260 units per mg of protein; Sigma), 95 ,g of MgCl2 in a total vol of 0.1 ml of phosphate buffer (0.1 M) (pH 7.4). The reaction was initiated by the *O...