guanosine-triphosphate and isocitric-acid

Side-chain cleavage (SCC) of endogenous cholesterol in adrenal mitochondria isolated from ACTH-treated rats indicates that the size of the reactive cholesterol pool depends on the reducing precursor. At optimal concentrations of reductant, this pool was typically at least 2 times greater for isocitrate than for succinate. Succinate-supported reactions were rapidly completed, were highly sensitive to a 2-min preincubation, and failed to deplete spectrally detected P-450SCC-cholesterol complexes. Cholesterol SCC with 1 mM isocitrate exhibited 2-3 times more fast-phase metabolism, a pronounced slow phase, insensitivity to preincubation, and 60% depletion of spectrally detected cholesterol-P-450SCC complexes. Addition of bovine serum albumin (BSA) and EDTA, either during homogenization or directly to the incubation, prevented preincubation losses in response to succinate and removed most of the difference between succinate and isocitrate activities. This effect of BSA/EDTA was reversed within 5 min by octanoate by a mechanism that was enhanced by Ca2+. These distinct reductant characteristics suggest that only a subpopulation of mitochondria or of pools of activity within individual mitochondria can support cholesterol SCC with succinate while isocitrate is necessary for the remainder. The rapid responses of succinate-supported metabolism to preincubation or to octanoate suggest depletion of a critical factor for cholesterol metabolism. Metabolism of added 20 alpha-hydroxycholesterol or deoxycorticosterone established that NADPH remained fully available after succinate-supported cholesterol metabolism had stopped or after preincubation. Cessation of pregnenolone formation, therefore, results from a failure to supply cholesterol, not inadequate NADPH. The preincubation effect suggests loss of an energy-dependent component that enhances this supply of cholesterol. One possibility tested was that GTP, an activator of intermembrane cholesterol transfer (Xu et al. (1989) J. Biol. Chem. 264, 17674-17680), was being lost. Added GTP slightly activated succinate-supported pregnenolone production but did not prevent preincubation-induced losses. alpha-Ketoglutarate, which can generate matrix GTP, is an effective reductant that, in combination with succinate, prevents preincubation-induced losses.

Topics: Adrenal Glands; Adrenocorticotropic Hormone; Animals; Calcium; Caprylates; Cholesterol; Cholesterol Side-Chain Cleavage Enzyme; Edetic Acid; Guanosine Triphosphate; Hydroxycholesterols; Isocitrates; Kinetics; Mitochondria; NADP; Pregnenolone; Rats; Serum Albumin, Bovine; Succinates; Succinic Acid

Previous studies from other laboratories, using rabbit reticulocyte lysate filtered through Sephadex G-25 or G-50, have demonstrated that glucose 6-phosphate is required to maintain active rates of translation, but its mechanism of action is currently unsettled. We have tested whether glucose 6-phosphate is required to prevent activation of the hemin-controlled translational repressor and the phosphorylation of the smallest or alpha subunit of eukaryotic initiation factor 2 (eIF-2). We have found that antibody to the hemin-controlled translational repressor can completely restore protein synthesis in reticulocyte lysate, filtered through Sephadex G-25, that is incubated in the absence of hemin and presence of glucose 6-phosphate, but cannot restore protein synthesis in such lysate incubated in the presence of hemin and absence of glucose 6-phosphate. We have also found, using a modification of the method of Matts and London [1984) J. Biol. Chem. 259, 6708-6711) to measure the ability of gel-filtered lysate to dissociate and exchange GDP from eIF-2.GDP, that this endogenous eIF-2B activity is reduced to the same low level in the presence of hemin and absence of glucose 6-phosphate as it is in the absence of hemin and presence of glucose 6-phosphate. Although there is a low level of phosphorylation of eIF-2 alpha in gel-filtered lysate given hemin but no glucose 6-phosphate, it cannot account for the loss of eIF-2B activity, since this phosphorylation is removed by antibody to the hemin-controlled translational repressor or isocitrate, which do not restore protein synthesis or eIF-2B activity, and not by fructose 1,6-diphosphate, which does partially restore protein synthesis and eIF-2B activity. These findings suggest that sugar phosphates may exert a direct effect on eIF-2B and may be required for its proper function. Additional support for this conclusion is our finding that protein synthesis and eIF-2B activity in partially hemin-deficient lysate can be restored by high levels of glucose 6-phosphate or fructose 1,6-diphosphate without a reduction in the level of phosphorylated eIF-2 alpha, suggesting that such levels of sugar phosphate may permit restoration of normal function with a limiting amount of eIF-2B.

Topics: Animals; Chromatography, Gel; Cyclic AMP; Cycloheximide; eIF-2 Kinase; Eukaryotic Initiation Factor-2; Fructosediphosphates; Glucose-6-Phosphate; Glucosephosphates; Guanosine Diphosphate; Guanosine Triphosphate; Hemin; Immunoglobulin G; Isocitrates; Kinetics; Peptide Initiation Factors; Phosphorylation; Protein Biosynthesis; Protein Kinases; Protein Synthesis Inhibitors; Proteins; Rabbits; Reticulocytes