Effect of triarimol on cholesterol biosynthesis in rat-liver subcellular fractions

Cholesterol biosynthesis was studied in rat-liver subcellular fractions incubated with DL-[2-¹⁴C]mevalonic acid in the presence and absence of triarimol (α-(2,4-dichlorophenyl)-α-phenyl-5-pyrimidine methanol). Triarimol strongly inhibits incorporation of radioactivity into cholesterol and this results in a large accumulation of radioactive lanosterol and 24,25-dihydro-lanosterol. The inhibition of lanosterol 14α-demethylase by triarimol was confirmed by assay of the enzyme in rat-liver microsomal fraction in the presence and absence of the inhibitor. Apart from a slight inhibition of Δ⁷-sterol-Δ⁵-dehydrogenase, triarimol did not affect the activity of any other enzyme involved in cholesterol biosynthesis from acetate.     

The distribution of free amino acids in subcellular fractions of guinea-pig brain

1. The free amino acids of homogenates of guinea-pig brain in 0.32m-sucrose and of subcellular fractions derived therefrom have been estimated by the method of Moore & Stein. 2. Seven amino acids together accounted for over 80% of the free amino compounds; these are, in decreasing order of abundance: glutamate, aspartate, gamma-aminobutyrate, glycine, serine, alanine and threonine. In addition, there are appreciable quantities of amide (presumably glutamine). 3. Control experiments showed that the pattern of free amino acid occurrence in sucrose homogenates was similar to that of brains of animals killed by freezing in liquid nitrogen and extracted immediately without thawing. 4. The subcellular distribution of the amino acids resembled that of soluble cytoplasmic markers; there was no specific localization in a fraction rich in isolated presynaptic nerve terminals of amino acids capable of exciting or depressing central neurones. 5. The significance of the results is discussed in relation to the possible role of centrally active amino acids as transmitters.     

Oxidative phosphorylation accompanying oxidation of short-chain fatty acids by rat-liver mitochondria

1. The factors concerned in the estimation of P/O ratios when fatty acids are oxidized by rat-liver mitochondria have been assessed. 2. The oxidation of butyrate, hexanoate and octanoate is accompanied by ATP synthesis. At low concentrations of the fatty acids, P/O ratios approximately 2.5 are obtained. 3. Oxidative phosphorylation is uncoupled, respiratory control ratios are lowered and respiration is inhibited when the concentration of the fatty acid in the incubating medium is raised (to 5-10mm); octanoate is a more potent uncoupler than either hexanoate or butyrate. 4. Serum albumin and carnitine, either singly or in combination, protect the mitochondria from the effect exerted by the fatty acids. 5. The rate of oxidation of short-chain fatty acids in the presence of ADP is increased in the presence of carnitine.     

Further studies on pyruvoyl amino acids

Pyruvoyl-α-aminoisobutyric acid was prepared, and its absorption characteristics in the ultraviolet compared with those of pyruvoylglycine, pyruvoyl-dl-alanine, and pyruvoyl-dl-phenylalanine. All four compounds in aqueous solution possess the same type of absorption, with maxima at 242–245 mμ and at 310–337 mμ. On alkalinization of the solution, the characteristic absorption of the latter three compounds disappears, and is not restored on acidification. On the other hand, the characteristic absorption of pyruvoyl-α-aminoisobutyric acid changes very little on alkalinization of the solution, and the small change is completely reversed on acidification.These differences have been interpreted as being due to the presence of at least one hydrogen atom on the α-carbon of the amino acid residue in pyruvoylglycine, pyruvoylalanine, and pyruvoylphenylalanine which permits ring closure in alkaline solution to the corresponding γ-hydroxy-pyrrolidonecarboxylic acid derivatives. Where no such hydrogen exists, as in pyruvoyl-α-aminoisobutyric acid, ring closure cannot occur, and the original starting material can be recovered.     

Effects of electroconvulsive shock and cycloheximide on the incorporation of amino acids into proteins of mouse brain subcellular fractions.

—The incorporation of [4,5-³H]lysine and [1-¹⁴C]leucine into the proteins of subcellular fractions of mouse brain was examined following a single electroconvulsive shock (ECS) or following cycloheximide injections. When the [³H]lysine was injected intraperitoneally immediately after the ECS the incorporation into total brain proteins was decreased by more than 50% as compared to sham controls. The proportion of lysine incorporated into the microsomal fraction was increased, but no changes were observed in the other subcellular fractions including the synaptosomal fraction. With extended pulses administered at various times after the ECS there was no change in total incorporation nor were selective effects seen in any subcellular fractions. With intracranial injections of both [³H]lysine and [¹⁴C]leucine the decreased incorporation caused by ECS was not observed, neither were there selective changes in any subcellular fraction. This lack of inhibition occurred because the intracranial injection itself severely inhibited [³H]lysine incorporation. Cycloheximide (30 mg/kg) which depressed [³H]lysine incorporation into brain proteins by 84% caused a selective depression of the incorporation into the cell-sap fraction and selective elevations into the microsomal and synaptosomal fractions. Similar changes were seen with a higher (amnestic) dose of cycloheximide (150 mg/kg) which inhibited incorporation by 94%. These data are interpreted in terms of the diverse mechanisms by which ECS and cycloheximide inhibit protein synthesis.