PURIFICATION AND CHARACTERIZATION OF FATTY-ACID SYNTHASE FROM CANDIDA-ALBICANS STRAIN-4918 AND 2 DERIVED SPONTANEOUS CERULENIN-RESISTANT MUTANTS

Fatty acid synthase from three strains of Candida albicans (parental s train 4918, and two spontaneous cerulenin-resistant mutants, 4918-2 an d 4918-10) has been purified and characterized. In all three cases the purification protocol included ammonium sulfate precipitation, fracti onation with butyl-Toyopearl, differential centrifugation and sediment ation velocity centrifugation. Inclusion of protease inhibitors, aprot inin, leupeptin and pepstatin was a prerequisite to maximize recoverie s. Polyacrylamide gel electrophoresis analysis demonstrated protocol e fficacy and showed the apparent molecular mass of the two enzyme sub-u nits from each strain to be 195 kDa and 210 kDa. The K-m (malonyl-CoA) and V-max of each fatty acid synthase were similar. In contrast, inac tivation kinetics of the respective enzymes in the presence of cerulen in showed enzyme activity from both mutants to differ significantly fr om the parent and from each other. Other experiments suggested in vivo cerulenin resistance of mutant strains is not solely attributable to enzyme alteration.