Mg2+-activated adenosine triphosphatase from Crithidia fasciculata: purification and inhibition by suramin and efrapeptin.

The mitochondrial Mg2+-activated adenosine triphosphatase (ATPase; EC 3.6.1.4) from the insect flagellate Crithidia fasciculata ATCC 11745 has been extracted from the membrane by chloroform treatment and purified to electrophoretic homogeneity by a method involving ammonium sulphate fractionation, gel filtration on Sephadex G-200 and DEAE-cellulose chromatography. The molecular weight of the native enzyme, determined by gel filtration, was about 350 000. Five subunits were detected by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate, with molecular weights of 54 000, 45 000, 35 000, 20 000 and 10 000. The membrane-bound, but not the soluble (F1) ATPase was inhibited by oligomycin and leucinostatin. Both forms of the enzyme were strongly inhibited by the antibiotic efrapeptin and the trypanocidal drug suramin. The inhibition by efrapeptin was of the mixed type, with double-reciprocal plots intersecting below the abscissa, as in the case of the enzyme present in beef heart submitochondrial particles. Suramin, on the other hand, acted as a non-competitive inhibitor of the membrane-bound ATPase and as a strictly competitive inhibitor of the purified F1 ATPase.

The citrate synthase from Bacillus Stearothermophilus.

Citrate synthase has been purified to apparent homogeneity from Bacillus stearothermophilus. Its kinetic and regulatory properties, and molecular weight, are similar to those of the enzymes from suitable mesophilic counterparts, but its thermal stability is considerably greater.

Purification and some properties of the citrate synthase from a marine Pseudomonas.

Citrate synthase (citrate-oxaloacetate lyase (CoA acetylating), EC 4.1.3.7) has been purified to electrophoretic homogeneity from a marine Pseudomonas. The enzyme was made up of identical subunits, with a molecular wieght of about 53 000, as determined by sodium dodecyl sulphate - polyacrylamide gel electrophoresis. The native enzyme (citrate synthase II, CS II) could be dissociated by dialysis against 20 mM phosphate (Pi), pH 7; the enzyme thus obtained (citrate synthase I, CS I) was still active, but presented different molecular weight and kinetic and regulatory properties. CS II was activated by adenosine monophosphate (AMP), Pi, and KCl, and inhibited by reduced nicotinamide adenine dinucleotide (NADH), being apparently insensitive to adenosine triphosphate (ATP) and adenosine diphosphate (ADP). The inhibition by NADH was completely counteracted by 0.1 mM AMP, but not by 50 mM Pi or 0.1 M KCl. The activation by KCl and Pi, or by KCl and AMP was nearly additive, whereas that by AMP and Pi was not. The activators acted essentially by increasing Vmax, although they also caused a decrease in the Km values. CS I was inhibited by ATP, ADP, AMP, and KCl, and was insensitive to NADH. CS I could be reassociated after elimination of Pi by dialysis, regaining the higher molecular weight and the activation by AMP characteristic of CS II.

[Regulation of citrate synthese in bacteria: Comparison of the action of various effectors on the enzymes of Rhodospirillum rurbum and Bacillus stearothermophilus]. / Regulacion de la citrato sintasa en bacterias: comparacion de la accion de varios efectores sobre las enzimas de rhodospirillum rubrum y bacillus stearothermophilus

A comparative study of the citrate synthases purified from the facultatively photosynthetic bacterium Rhodospirillum rubrum (Gram negative) and the thermophile Bacillus stearothermophilus (Gram positive) was made. The citrate synthase from R. rubrum was activated by KCl (6-fold at 0.1 M KCl) and, less effectively, by NaCl and NH4Cl. Its molecular weight was about 300,000. The enzyme was strongly inhibited by NADH, and this inhibition was counteracted by AMP. The citrate synthase from B. stearothermophilus was little affected by KCl, NaCl and NH4Cl, all of which activated by about 25% at 0.1 M. Its molecular weight was ca 100,000. The enzyme was not affected by NADH or AMP. Both citrate synthases were insensitive to alpah-oxoglutarate concentrations up to 5 mM, and were inhibited by ATP; the B. stearothermophilus enzyme was more strongly inhibited than the R. rubrum enzyme. In both cases the ATP inhibition was strictly competitive towards acetyl-CoA and non-competitive towards oxaloacetate. Both enzymes, in spite of the peculiar physiological properties of their bacterial sources, followed the close correlation between the properties of the citrate synthase and the taxonomical position of the microorganism, proposed by Weitzman and his co-workers.

Regulatory properties of the citrate synthase from Rhodospirillum rubrum.

Citrate synthase, purified 600-fold from Rhodospirillum rubrum, is activated by KCl and inhibited by ATP and NADH; the effect of the latter inhibitor is completely counteracted by AMP and partially counteracted by KCl.

NAD-linked glutamate dehydrogenase in Trypanosoma cruzi.

1. Epimastigotes of Trypanosoma cruzi, Tulahuén strain, contained a NAD-linked glutamate dehydrogenase (EC 1.4.1.3), in addition to the already known NADP-linked enzyme enzyme (EC 1.4.1.4). 2. The partially purified NAD-linked enzyme had a higher molecular weight and was much more labile than the NADP-linked enzyme, and was inhibited by purine nucleotides. 3. These results further emphasize the difference in glutamate metabolism between the parasite and its mammalian host.

CO2-fixing enzymes in Pseudomonas fluorescens.

Pseudomonas fluorescens grown on glucose or glutamate at 1 or 20 degrees C, or on acetate at 20 degrees C, as sole carbon sources, contained both pyruvate carboxylase and phosphoenolpyruvate carboxylase. Pyruvate carboxylase was insensitive to acetyl-coenzyme A and L-aspartate, and its level in cell-free extracts was markedly dependent on the carbon source for growth, the highest specific activity being attained in glucose-grown cells. Phosphoenolpyruvate carboxylase, on the other hand, although less dependent on the nature of the carbon source,showed its highest level in acetate-grown cells; the enzyme activity required acetyl-coenzyme A and was strongly inhibited by L-aspartate. The micro-organism had, in addition, a phosphoenolpyruvate carboxykinase, which showed its highest specific activity in cells grown on acetate, and a NADP-linked malate enzyme, apparently repressed by acetate and showing its highest specific activity in glutamate-grown cells.

[Metabolism of a psychrophilic bacterium from fresh water]. / Metabolismo de una bacteria criófila de agua dulce

Psychophilic microorganisms, able to grow at 0-1 degrees C, were isolated from water obtained from the Paraná River at Rosario. One of the strains, R-12, was identified as Pseudomonas fluorescens according to the description in Bergey's Manual of Determinative Bacteriology (8th Ed., 1974). The microorganism was able to grow in liquid minimal medium with glucose, acetate, glutamate or casein hydrolysate as sole carbon source, at 20 degrees C. The enzymes of the Entner-Doudoroff pathway were induced in cells grown on glucose. The Krebs cycle was apparently operative in all cases; the lower levels of citrate synthase and isocitrate dehydrogenase were found in glucose-grown cells. Isocitrate lyase was present at a high concentration, and malate synthase considerably increased, in acetate-grown cells, thus suggesting the operation of the glyoxylate cycle. When cells were grown on glucose the anaplerotic function was probably fulfilled by pyruvate carboxylase, although phosphoenolpyruvate carboxylase was also present. The gluconeogenic enzyme phosphoenolpyruvate carboxykinase was repressed by glucose; malic enzyme was repressed by acetate. The regulatory patterns shown by citrate synthase and pyruvate carboxylase were similar to those described for the enzymes from other Pseudomonas. Whole cells of the R-12 strain were able to decarboxylate the aminoacids serine, aspartate and glutamate. Aspartate aminotransferase was present at high levels in aminoacid-grown cells, thus suggesting a catabolic role, whereas glutamate dehydrogenase had increased levels in glucose - or acetate-grown cells, suggesting that it fulfilled a mainly biosynthetic role.