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 Mr
45000 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 μm and the Vmax at
12 mM AMC released min−1 mg−1
of protein for L-Leu-AMC. Since this natural peptidase is sensitive
to the protease inhibitors 1,10-phenantroline
and ethylenediaminetetraacetic acid, it is considered as a metallopeptidase.