Abstract
This paper describes isolation of electrophoretically homogenous enolase from Candida albicans. The purification involved: disintegration of C. albicans cells in a Braun’s mill (67–100%) ammonium sulfate precipitation, chromatography on DEAE-Sephadex A-50 at pH 9.0 and chromatography on CM-Sephadex A-50 at pH 6.2. The procedure resulted in a 30-fold purification of the enzyme with a recovery rate of 6% and a specific activity 35 U mg −1. The subunit molecular weight was 46 kDa and the pH optimum of the enzyme was 6.8. K m and V max values for the 2PGA→PEP reaction were determined ( K m=0.95 mM, V max=4200 μmol min −1 μmol −1). In the absence of orthophosphate, inhibition by fluoride was competitive, which became noncompetitive in the presence of phosphate. It was confirmed that Mg 2+ is the most potent activator ( K m=0.286 mM); Mn 2+ gave less activity and Zn 2+ less still. It was also demonstrated that the presence of two types of cations in the reaction mixture nullified the activatory effect of the stronger agent. Properties of the enzyme from C. albicans are compared with those of enolases from other sources.
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