1887

Abstract

An inorganic pyrophosphatase was purified from strain BERRE SOL. Its activity was increased up to 130-fold by reduction with reagents (NaSO, Na[BH], NaS, 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 O, N 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-CoCl as co-factor; with 3 mM-MgCl or 0·3 mM-MnCl 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.

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/content/journal/micro/10.1099/00221287-67-2-145
1971-08-01
2020-11-30
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http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-67-2-145
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