- Volume 67, Issue 2, 1971

An inorganic pyrophosphatase was purified from Desulfovibrio desulfuricans strain BERRE SOL. Its activity was increased up to 130-fold by reduction with reagents (Na2S2O4, Na[BH4], Na2S, etc.) of values less than - 0·14V at pH 7·0 and 25°. The pure enzyme was isoelectric at pH 6·55; its molecular weight was 41,600 according to amino acid analysis, consistent with values of 43,000 ± 7000 from ultracentrifugation and exclusion chromatography. The enzyme was very unstable at both 4° and 15° and was easily damaged physically: it lost 75% of activity on freezing and thawing and was progressively destroyed if O2, N2 or CO were bubbled through its solution. With 6·25 mM-sodium pyrophosphate, the enzyme showed maximum activity at pH 6·2 with 1 mM-CoCl2 as co-factor; with 3 mM-MgCl2 or 0·3 mM-MnCl2 the pH optimum was 8·0. The molecular weight and isoelectric point of the form active without reduction were the same as those of the form active only after reduction; no gross conformational change on reductant activation was detected by spectropolarimetry and fluorescence tests, but reductant activation was associated with an increase in the number of -SH groups in the molecule and a change in electrophoretic mobility at pH 10.

Neither crude nor highly purified enzyme preparations were sensitive to oxygen but the degree of reductant activation of the extracted enzyme depended on the extent to which the intact bacteria had been exposed to oxygen. When bacteria from a continuous culture were harvested with minimum exposure to air they contained active enzyme showing little reductant activation; comparable bacteria resuspended in environments containing dissolved oxygen, but neither a carbon source nor sulphate, yielded enzyme preparations which were almost or completely inactive without a reductant. Addition of sodium lactate and sulphate to the aerated bacteria reversed the phenomenon: the enzyme when extracted was active without reductant. Intracellular inactivation and reactivation processes were rapid and were not influenced by chloramphenicol; alteration of the nutritional status of the population did not affect the intracellular state of the enzyme. Reversible inactivation of inorganic pyrophosphatase was observed in four other strains; it may be a survival mechanism in Desulfovibrio, and in Clostridia, which enables such anaerobes to conserve ATP while in aerobic environments.

Incorporation of radioactive glucose and N-acetyl glucosamine into the walls of growing fungal hyphae has been studied using light microscopic autoradiography. Autoradiographs of Phytophthora parasitica with glucose as substrate and Neurospora crassa and Schizophyllum commune with glucose or N-acetyl glucosamine as substrate show that most of the incorporation after incubation times as short as 1 min. is at the extreme hyphal tip. N-Acetyl glucosamine is incorporated less into the subapical region than glucose, and N. crassa shows less subapical incorporation of both substrates than the other two fungi.

The lemmas and paleas of individual barley grains were detached and the amount of fungal mycelium present was determined by direct microscopic observation. The area in which mycelium was present varied from 19·7 to 87·7% of the lemma, and from 44·2 to 92·8% of the palea. The total estimated length of mycelium observed varied from 19 to 177 cm. in individual grains. It is suggested that these lengths represented dry weights of mycelium of the order of 1·5 to 1·9 μg. in individual grains. The indirect fluorescent antibody technique was used to determine the extent of Penicillium cyclopium mycelium. The area in which this species, predominant in plate cultures, was present did not exceed 22·7% of the lemma, and no more than 17·2% of the palea. The results indicated that this technique is useful in the detection and estimation of individual species in barley grains and that it might be of use in other plant disease studies.

Homogenates of Tetrahymena pyriformis suspensions were prepared aftergrowth in the absence and presence of chloramphenicol (500 μg./ml.). Sedimentation of organelles through gradients of aqueous sucrose solutions was studied by means of a zonal centrifuge. Mitochondria (marker enzyme, malate dehydrogenase) from organisms grown with chloramphenicol sediment more slowly (median sedimentation coefficient in water at 20° = 10,600 S) than those from normal cells (median sedimentation coefficient in water at 20° = 90,000 S, which confirms that the former are of smaller volume than normal mitochondria. After high-speed zonal centrifugation both mitochondria (marker enzymes, NADH and NADPH-cytochrome c oxidoreductases) attain similar equilibrium densities (median density p = 1·21 to 1·22). The buoyant density (median p = 1·23) of peroxisomes (marker enzyme, catalase) is not altered by the inclusion of chloramphenicol in the growth medium, but the proportion of non-sedimentable catalase is increased. Chloramphenicol-inhibited growth also leads to a change in the distribution of lysosomes (marker enzymes, acid p-nitrophenolphosphatase, and N-acetyl-β(D)-glucosaminidase); a marked reduction in the population at p = 1·15 to 1·20 is accompanied by the appearance at p= 1·09 to 1·15 of a new class of organelles containing acid hydrolases.

The physiological, morphological and serological properties of 47 species of 13 genera, excluding Saccharomyces, were subjected to numerical analysis. Fifteen groups were distinguished within the genera Brettanomyces, Debaryomyces, Hanseniaspora, Hansenula, Kloeckera, Nadsonia, Pachysolen, Pichia, Saccharomy-codes, Schizosaccharomyces and Schwanniomyces and species of Candida and Torulopsis which were not the imperfect forms of Saccharomyces species. There was no simple correlation between taxonomic groupings and serological properties, but by a combination of tests - anaerobic growth, morphology, serology and growth on lysine and nitrate as sole source of nitrogen - the various groups were easily distinguished.