Jean-Louis Jacquemin

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.

Detection and Immunolocalization of Human Erythrocyte Spectrin Immunoanalogues in Toxoplasma gondii (Protozoan, Parasite)

ABSTRACT. We demonstrated here the presence of proteins antigenically related to human erythroid spectrin in the parasitic protozoan Toxoplasma gondii. A high molecular weight doublet (M, 245‐240,000), present in equimolar ratio, and low molecular weight polypeptides (M, 75,000) were reacted with monoclonal and polyclonal anti‐human erythroid spectrin antibodies on electroblotted nitrocellulose sheets. Indirect immunofluorescence assay clearly showed that these proteins were localized in the anterior pole of the organism. Immunogold staining further revealed specific labeling of conoid, rhoptries, micronemes, and dense granules of the apical complex. The presence of the M, 245–240,000 doublet and the M, 75,000 spectrin‐like proteins in the anterior pole of T. gondii may probably be consistent with a structural stabilizer function in its organciles which are suspected to be involved in the process of host cell invasion.

Detection of Galactomannan for Diagnosis of Fungal Rhinosinusitis

Fungus ball of the paranasal sinus is the growth of a fungal mass (mycetoma) within the sinus cavity, usually caused by molds; endoscopic surgery is the usual treatment ([2][1], [4][2]). Because of the poor viability of the fungi, diagnosis based on fungal cultures of nasal secretion or material

Degradation of host components by a metallopeptidase of Cryptococcus neoformans

Proteolytic activities were detected in a cytosoluble 100 000 g extract of Cryptococcus neoformans using 7-amido-4-methylcoumarin substrates. The main proteolytic activity cleaved the L-arginine-AMC substrate. This protease was purified by high performance liquid chromatography and had a molecular weight of 80 kDa as demonstrated by SDS-PAGE in the presence of reducing conditions. The enzyme was inhibited by EDTA and o -phenanthroline which are metallopeptidase inhibitors. The protease displayed high enzymatic activity between pH 5.8 and 7, and its pl was localised at pH 5.04. Furthermore, this metallopeptidase was not secreted in culture supernatants, but was able to degrade partially or totally some of the extracellular matrix components, as fibronectin or laminin, and host lgG.

Purification of an intracellular metallopeptidase of Mr 45000 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 μM and the V max 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.