Purification and characterisation of a metallopeptidase of Candida albicans.
A novel aminopeptidase was purified by high performance liquid chromatography from a cytosoluble 100,000 g extract of Candida albicans on the basis of its ability to cleave L-arginine 7-amino-4-methylcoumarin. The purification factor was 36 and the yield was 20%. The native enzyme had a mol. wt of 52 kDa as demonstrated by SDS-PAGE in the presence or absence of reducing conditions and exhibited an iso-electric point of 4.3. The aminopeptidase showed optimum activity at pH 7.2, a Michaelis constant of c. 50 microM and a Vmax at 19 mM AMC released/min/mg of protein for L-Arg-AMC. This enzyme was shown to cleave at low affinity L-leucine-7-amino-4-methylcoumarin as demonstrated by the spectrofluorimetric method. The enzyme was strongly inhibited by specific metallo-enzyme inhibitors-EDTA and o-phenanthroline. Furthermore, there is evidence that a similar or identical enzyme occurs in other C. albicans clinical isolates and other Candida spp.
el Moudni
B
B
Laboratoire de Parasitologie et Mycologie Médicales, Centre Hospitalier Universitaire La Milétrie, Poitiers, France.
Rodier
M H
MH
Barrault
C
C
Ghazali
M
M
Jacquemin
J L
JL
D000626:Aminopeptidases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D002175:Candida / Q000201:enzymology; D002176:Candida albicans / Q000201:enzymology*; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D010618:Phenanthrolines / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity
D010618:Phenanthrolines; D011480:Protease Inhibitors; D004492:Edetic Acid; D000626:Aminopeptidases; D008666:Metalloendopeptidases; C025205:1,10-phenanthroline