Dehydroepiandrosterone (DHEA), an intermediate in the biosynthesis of testosterone and estrogens, exerts several physiological effects not involving the sex hormones. When fed to rats it induces the thermogenic enzymes mitochondrial sn-glycerol-3-phosphate dehydrogenase and cytosolic malic enzyme in their livers. Animals and humans, and their excised tissues, are known to hydroxylate DHEA at several positions and to interconvert 7a-hydroxy-DHEA, 7,8-hydroxy-DHEA, 7-oxo-DHEA, and the corresponding derivatives of androst-5-enediol. We report here that these 7-oxygenated derivatives are active inducers of these thermogenic enzymes in rats and that the 7-oxo derivatives are more active than the parent steroids. We postulate that the 7cr-hydroxy and 7-oxo derivatives are on a metabolic pathway from DHEA to more active steroid hormones. These 7-oxo steroids have potential as therapeutic agents because of their increased activity and because they are not convertible to either testosterone or estrogens.We wish to report the biological activity of some known and some newly discovered steroids structurally related to dehydroepiandrosterone (androst-5-ene-313-ol-17-one; DHEA). DHEA is produced in the adrenals and brain and is the most abundant steroid in the blood of adult humans; it reaches a maximum concentration at 20-25 years of age and declines thereafter. It circulates mainly as the sulfate ester but the ester and free steroid are metabolically interconverted (1, 2). DHEA is an intermediate in the metabolic conversion of cholesterol to testosterone and estrogens and it also exerts several physiological effects independent of the sex hormones. In relatively large doses, it causes weight loss in genetically obese (3) and normal (4) animals without affecting food intake; it depresses blood cholesterol levels in men (5), rats (6), and dogs (7); it decreases blood sugar concentration in diabetic mice (8); it enhances resistance of mice to viral infections (9, 10); it reduces the incidence of spontaneous (11, 12) and carcinogen-induced (13) tumors in mice; and it improves memory in aged mice (14). Body weight responses in humans treated with DHEA are questionable (5,(15)(16)(17)(18)(19).The diversity of the responses to DHEA and the large doses (-0.5% of the diet for animals) required to elicit most of them led to the postulate that DHEA might be the precursor of steroid hormones other than the sex hormones and that these metabolites might be active in smaller doses and might display specificity for one, or only a few, of the effects described above.We therefore synthesized known and possible metabolites of DHEA in the hope of finding compounds with greater biological activity. Another objective was to find metabolites of DHEA that retained the activities of the parent except for the ability to form androgens and estrogens. DHEA should not be used by women for extended periods because it increases their circulating testosterone and dihydrotestosterone manyfoldThe publication costs of this article were defrayed in p...

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The activation and catalytic mechanism of corn mitochondrial F, were examined for the two distinct forms of the enzyme which appear upon storage in ammonium sulfate or glycerol. Apparently irreversible differences in the stability of the two active forms were found. Nucleosidetriphosphate induced activation of the enzyme was found to produce lasting effects on subsequent catalysis. These effects varied with both the nucleotide used for activation, and the hydrolyzed species. The substrate and metal specificity were examined with the ATP activated enzyme. Mg2 and Ca24 were found to be the most effective at promoting ATP hydrolysis. erties of the ATPases which catalyze the committed step in each pathway.In an accompanying study ofthis series, a method of obtaining purified corn mitochondrial F, was described and the subunit structure and immunochemical properties examined (18). In the current study, the activation and catalytic properties of the enzyme are examined. MATERIALS AND METHODSCorn mitochondrial F,, purified as described in the accompanying manuscript (18), was stored in 4 mm ATP, 1 mm EDTA (pH 7.0) either as a 50% (NH4)2SO4 slurry at 4°C or in 25% glycerol at -70C.Following a centrifugation and rinse step, the (NH4)2SO4 preparation was dissolved in an activation buffer consisting of 50 mM Tricine-NaOH, 0.1 mM EDTA (pH 8.0) and, unless otherwise specified, 5 mm ATP. The glycerol-stored enzyme was prepared by passage through a centrifuge column consisting of a l-ml plastic pipette tip packed with Bio-Rad P-6DG desalting gel and equilibrated with the activation buffer.In experiments dealing with NTP activation of the enzyme, activation time was measured relative to the time of dissolution or centrifugation into the activation buffer. All other assays were performed on enzyme that had been activated by incubation at 25°C for at least 1 h in the activation buffer with 5 mm NTP. Coupled assays of ATPase activity were performed at 30'C in a system consisting of 50 mM Tricine-NaOH (pH 8.0), 1.0 mm KCl, 2 mm phosphoenolpyruvate, 0.3 mM NADH, 12 units of pyruvate kinase, and 17 units lactate dehydrogenase in a total volume of 1 ml. Phosphate assays were performed in 50 mm Tricine-NaOH (pH 8.0 and 30C) and phosphate determined according to the method of Heinonen and Lahti (9). Protein concentrations were determined according to Bradford (3), using the Bio-Rad dye concentrate and fraction V BSA as a standard. Except as specified, all reagents were from Sigma. RESULTSA number of properties of the corn mitochondrial F,-ATPase were found to depend on whether the purified enzyme was stored as an (NH4)2S04 precipitate, or frozen in 25% glycerol. The ATPase activity of the glycerol-stored enzyme was generally higher and less subject to decay than the (NH4).SO4-stored material. While the former retained an activity of up to 8 Amol min-' mg-' (at 5 mm ATP) for 2 months, the activity of the precipitated enzyme decayed from -3 to 0.5 over the same period. Kinetic analyses ofan (NH4)2S04-stored preparation over this ...

Corn mitochondrial F1-ATPase was purified from submitochondrial particles by chloroform extraction. Enzyme stored in ammonium sulfate at 40C was substantially activated by ATP, while enzyme stored at -70'C in 25% glycerol was not. Enzyme in glycerol remained fully active (8)(9) micromoles Pi released per minute per milligram), while the ammonium sulfate preparations steadily lost activity over a 2-month storage period. The enzyme was cold labile, and inactived by 4 minutes at 60'C. Treatment with octylglucoside resulted in complete loss of activity, while vanadate had no effect on activity. The apparent subunit molecular weights of corn mitochondrial F,-ATPase were determined by SDSpolyacrylamide gel electrophoresis to be 58,000 (a), 55,000 (8), 35,000 (-y), 22,000 (a), and 12,000 (e). Monoclonal (9). No detailed comparisons of mitochondrial F,-ATPase and chloroplastic CF,-ATPase from the same plant species has as yet been accomplished, and the similarities or differences between these enzymes can only be inferred by studies ofanimal mitochondrial Fr-ATPases.The fact that plant cells contain two unique organelles capable of energy production by oxidative phosphorylation in one case, or photosynthetic phosphorylation in the other, makes them an attractive model for comparative studies. Detailed studies of plant mitochondrial F,-ATPase will provide the basis for comparison to chloroplastic CF,-ATPase. Comparisons of these two enzymes from the same cell may provide valuable information on aspects of structure, regulation, biosynthesis, and evolution of the enzyme within these two organelles. This paper reports the isolation, purification, physical and enzymic properties of the F,-ATPase from corn mitochondria. In addition, the antigenic properties ofthis enzyme are compared to those from CF,-ATPases and mamallian and E. coli F,-ATPases using both polyclonal and monoclonal antibodies. MATERIALS AND METHODS Purification of