Sulphite: cytochrome c oxidoreductase (sulphite dehydrogenase) was purified 2000-fold from Thiobacillus versutus (A2). The enzyme monomer had a molecular weight of 44000 and a pI value of 4·5 · 0·3. Cytochrome c 551 was intimately associated with the enzyme: separation of the two greatly decreased sulphite dehydrogenase activity, which was not restored by remixing them. The enzyme had a pH optimum around pH 8·0, exhibited a K m of 14 μM for sulphite, and was inhibited noncompetitively by phosphate, with a K i value of 12 mM. It was also inhibited by p-hydroxymercuribenzoate and cyanide. Its involvement in the oxidation of thiosulphate in T. versutus is discussed.
The effect of various nitrogen sources on lipid accumulation by 17 species and strains of yeast was examined. Organic nitrogen sources resulted in considerably increased lipid contents only in strains of Rhodosporidium toruloides. Lipid accumulation in Rs. toruloides CBS 14 increased from 18% (w/w), with NH4Cl as nitrogen source, to above 50% (w/w) when glutamate, urea or arginine was used. Stimulation of lipid production by glutamate was not observed when the yeast was grown in continuous culture with nitrogen-limiting medium. The increase in lipid content of glutamate-grown cells in batch culture was accompanied by a marked increase in the intracellular citrate concentration and its excretion from the cells. The pattern of citrate accumulation in glutamate-grown cells was mirrored by the accumulation of other metabolites, especially 2-oxoglutarate and NH+ 4 ions, which were produced as a result of the increased catabolism of glutamate. It is proposed that the products of glutamate metabolism in Rs. toruloides play a major role in regulating the flux of carbon to precursors of lipid biosynthesis, such as citrate.
Rhodosporidium toruloides CBS 14 was grown in batch culture with urea as principal nitrogen source. The lipid content increased from 18% (w/w) with NH+ 4-grown cells to 52% (w/w) after 90 h growth. Urea was rapidly taken up and catabolized to release intracellular NH+ 4, which accumulated between 10 and 30 h growth. The increase in pool NH+ 4 content was mirrored by an increase in citric acid accumulation and excretion from the cells. The production of intracellular NH+ 4, sufficient to permit lipid accumulation, could be attributed to the increase in activity of urease over this period. Similarly, other catabolic enzymes, such as arginase, threonine dehydratase and NAD+: glutamate dehydrogenase, were also induced (or derepressed) when the respective amino acids were used as medium nitrogen source. Growth with mixed organic and inorganic nitrogen compounds considerably decreased the lipid content and was accompanied by a reduction in activity of the various catabolic enzymes concerned. The significance of nitrogen catabolism during lipid accumulation in this yeast is discussed.
Three binding proteins, one specific for guanosine 3′: 5′-monophosphate (cGMP) and two specific for adenosine 3′: 5′-monophosphate (CAMP) have been partially purified from vegetative cells of Myxococcus xanthus M300. The cGMP binding activity was found in the periplasmic shock fluid. Scatchard analysis indicated only a single class of binding sites with high affinity (apparent KD, 42 nM). The two cAMP binding activities were physically distinct, as indicated by their elution patterns from DEAE-cellulose, KD values and cellular locations. The cytoplasmic cAMP binding protein, which is probably identical to that previously isolated from developing myxospores of M. xanthus had an apparent KD of 57 nM, whereas the periplasmic cAMP binding protein had an apparent KD of 1 PM. During development, the nucleotide binding proteins exhibited changes in activities consistent with their postulated roles during fruiting body development.
Myxococcus xanthus M300 vegetative cells contained significant amounts of adenylate and guanylate cyclase activity. The latter was distributed between the 100000 g supernatant and pellet fractions, required divalent cations for activity and exhibited an apparent Km of 1 mm. Adenylate cyclase activity was detected both in the l00000 g supernatant and pellet. The supernatant enzyme had an apparent K, of 220 PM with a Hill coefficient of 1.9, whereas the pellet enzyme had a Km of 72 μM and a Hill coefficient of 1.0. The isoenzymes differed in their pH optima and divalent cation requirements for optimal activity. During development of Myxococcus xanthus, the nucleotide cyclase activities exhibited changes that were substantially consistent with the roles postulated for each in a previously proposed model.
Chitinase activity was detected in the supernatant fraction of a high-speed centrifugation preparation of broken Candida albicans yeast cells. The enzyme showed peak activity during the rapid budding phase of growth and was found to parallel the chitin synthase activity. The optimum conditions for the hydrolysis of chitin, regenerated from acetylation of chitosan, were determined. Analysis of the kinetics of the enzyme-substrate interaction and a measurement of their binding suggests that an equilibrium binding situation exists and that the kinetics follow a Langmuir isotherm interaction.