Purification of an intracellular metallopeptidase of M-r 45 000 in Fusarium moniliforme

Cytoplasmic extracts of Fusarium moniliforme contained a peptidase activity, able to cleave preferentially L-Leu-7-amino-4-methylcoumarin fluorogenic substrate. No activity towards this substrate was detected in various culture media of F. moniliforme supplemented or not with different substrates (bovine serum albumin bovine haemoglobin, collagen or gelatin), proving that the peptidase was not secreted in these conditions. The cytoplasmic enzyme was purified by high performance liquid chromatography using a combination of an anionic exchange and gel filtration columns. The purified activity gave a single band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis, estimated at M-r 45 000 under reducing conditions. The aminopeptidase showed an optimum activity at pH 7.2, an isoelectric point at 4.1, the Michaelis constant was at 50 mu M and the V-max at 12 mM AMC released min(-1) mg(-1) of protein for L-Leu-AR IC Since this natural peptidase is sensitive to the protease inhibitors 1,10-phenantroline and ethylenediaminetetraacetic add, it is considered as a metallopeptidase.