Biomedical subjects
Publications and source records attributed to B Cheng.
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Following 15-30 min exposure to monensin, adrenocorticotropic hormone (ACTH)-stimulated steroidogenesis in cultured adrenal cells is inhibited by 37-48%. Electron microscopic studies reveal that, in monensin-treated cells, the Golgi complexes are disrupted into large vacuolar structures with loss of its organized structure indicating that the action of monensin on the organelles is comparably rapid. The inhibition is fully reversed after removal of the monensin-containing medium and exposure to fresh growth medium for a subsequent 4-24 h prior to stimulation. Concomitant with the restoration of full steroidogenic activity, the disrupted organelles are extensively reorganized in the cells after exposure to fresh growth medium for 4-24 h. These findings, which demonstrate, for the first time, a correlation between the morphology of the Golgi complex and steroidogenic activity, strengthen the possibility that the organelle may be involved in the regulation of steroidogenesis.
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It has previously been shown that the steroidogenic action of adrenocorticotropic hormone (ACTH) is accompanied by characteristic alterations in cell ultrastructure. These include hypertrophy of the Golgi complex associated with increased vesicle formation and striking elevations of acid phosphatase activity in the Golgi complex and lysosomes. To investigate a possible relationship of these phenomena to steroidogenic function in monolayer cultures of murine adrenal tumor cells, monensin, a carboxylic ionophore which disrupts the ordered structure and transport function of the Golgi complex, was used. Monensin, at a concentration of 1.2 microM, causes massive vacuolization and hypertrophy of the Golgi complex. No effect on mitochondrial structure was seen. Monensin, 0.6-1.2 microM, inhibits both ACTH-stimulated and basal steroidogenesis by approximately 50% in incubations of 4 h or less. Dibutyryl-cAMP-stimulated steroidogenesis was inhibited to a similar degree. Incubations were carried out in serum-free media to eliminate possible effects due to exogenous cholesterol transport into the cell. There were no direct inhibitory effects of monensin on cholesterol side-chain cleavage (SCC) activity in isolated mitochondria. In contrast, mitochondria isolated from cells previously treated with monensin had a reduced capacity for this activity. These experiments suggest that monensin inhibits transport of cholesterol from the Golgi complex to the mitochondrial site of steroidogenesis action or interferes with the transport of key mitochondrial proteins synthesized on cytoplasmic ribosomes.
Inner and outer submitochondrial membranes were prepared after disruption of malate-treated bovine adrenocortical mitochondria. It was found that a part of the endogenous cholesterol in the inner membrane (approx. 50%) was rapidly utilized by the cholesterol side-chain cleavage reaction. The utilization of cholesterol in the outer membrane, on the other hand, was inefficient and slow in spite of the fact that cholesterol concentration is higher in the outer than in the inner membrane. When the inner membrane prepared from untreated mitochondria was incubated for 20 min in the presence of a reconstituted cytochrome P-450-reducing system, the inner membrane cholesterol was depleted by approximately 70%. The half-life of the depletion reaction was 2-3 min. In addition, when the outer membrane plus the soluble fraction from the untreated mitochondria were added as a source of cholesterol to the inner membrane fraction, a marginal increase in the production of steroids was observed. From these results it is concluded that a portion of the inner membrane cholesterol can be steroidogenic, whereas the rest of the cholesterol is non-steroidogenic.
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Glucose and anoxia accelerate the photocount due to luminescence of Ehrlich Ascites cells. Adriamycin also has this effect if glucose is present. Comparison with a chemical standard combined with estimates of cellular and population transmittance yield a photon generation rate of at least 10 . s-1 . cell-1 in the presence of 10(-2) M glucose, and twice this with anoxic conditions or 10(-5) M adriamycin. Effects of adriamycin on Ehrlich Ascites cell respiration may depend on the presence of glucose.
The cholesterol contents and phospholipid compositions of mitochondria, microsomes and submitochondrial membranes from bovine adrenal cortex have been analyzed quantitatively. From our results, the following cholesterol contents were obtained: mitochondria, 6.2 +/- 0.9 mol %; microsomes, 18.4 +/- 2.8 mol %; mitochondrial inner membrane, 2.8 +/- 0.6 mol %; and mitochondrial outer membrane, 8.3 +/- 1.3 mol %. In addition, the phospholipid compositions of the mitochondrial inner and outer membranes were determined for the first time. Cardiolipin was found to be enriched in the inner membrane, whereas phosphatidylinositol was richer in the outer membrane. The general features of phospholipid compositions in the submitochondrial membranes resembled that of rat liver mitochondria.
The pH-dependence of fumarylacetoacetase (4-fumarylacetoacetate fumaryl-hydrolase, EC 3.7.1.2) activity was studied in the pH range 6.25-8.50. After correction of the substrate concentration for enolate formation, the Michealis constant was found to be pH independent in this range. Likewise, the Ki values for the competitive inhibitors chloride and fluoride were found to be independent of pH between 6.25-8.50. A bell-shaped curve described the log V vs. pH dependence, and ionization constants of 6.5 and 8.2 were calculated. Tentatively an imidazole group and a sulfhydryl group were assigned to the constants 6.5 and 8.2, respectively. Both p-hydroxymercuribenzoate and 5,5'-dithiobis(2-nitrobenzoic acid) react with both sulfhydryl groups per subunit in the native protein and three sulfhydryl groups per subunit in the denatured protein. Substrate protects one sulfhydryl group in the native protein from reaction with 5,5'-dithiobis(2-nitrobenzoid acid). Substrate or the competitive inhibitor, fluoride, protect the enzyme from inactivation by p-hydroxymercuribenzoate. In addition p-hydroxymercuribenzoate shows saturation kinetics. Neither sulfhydryl inhibitor completely inactivates the enzyme. The enzyme is described as having three sulfhydryl groups per subunit, one of which is inaccessible to the sulfhydryl specific reagents when the protein is in the native state. One of the two accessible sulfhydryl groups is either near the active site or essential in maintaining the structure of the protein.
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