1887

Abstract

Two aminopeptidases, designated as aminopeptidase A and B, were purified 1720- and 1950-fold, respectively, from the culture filtrate of F by ammonium sulphate fractionation, a series of column chromatography steps on DEAE-Sephadex, DEAE-Trisacryl M and Mono-Q, and gel filtration on Superose-6. Aminopeptidase A accounted for 85% of the aminopeptidase activity remaining by the end of purification. The purified enzymes were homogeneous as judged by disc gel electrophoresis. Aminopeptidases A and B showed the same pH optimum of 9·3 and apparent temperature optimum of 40 °C, although the former showed slightly higher pH stability than the latter. NaCl or KC1 concentrations to 2 did not affect the activities or stabilities of aminopeptidases A and B. Both enzymes were completely inhibited by incubation with EDTA, indicating that they are metalloenzymes, and were reactivated by incubation with Ca, Co, Mg, Zn or Mn. The values of aminopeptidases A and B were estimated as 150000 and 110000, respectively, by gel filtration on Superose-6, and as 36000 and 26000, respectively, by SDS-PAGE. It was thus assumed that each of the native enzymes exists as a tetramer. The values of aminopeptidase A and B for -leucine--nitroanilide were calculated to be 16·1 and 15·2 m, respectively. The substrate specificities of aminopeptidases A and B were similar to each other.

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1989-07-01
2021-10-20
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