Substrate-dependent pH optima of gp63 purified from seven strains of Leishmania.
The major surface glycoprotein of Leishmania, gp63, is a membrane-bound metalloprotease. Contradictory data supporting a neutral or acidic nature of this enzyme have been presented. Seven strains of Old and New World Leishmania, including Leishmania donovani complex (Leishmania infantum and L. donovani), Leishmania major, Leishmania tropica and Leishmania mexicana amazonensis were used for the purification and comparative study of gp63. The protein was extracted from promastigotes by phase separation in Triton X-114 and purified by anion exchange chromatography. In agreement with previous reports, all purified gp63 were found to be structurally and immunologically related. Both membrane-bound gp63, on the surface of promastigotes, and the purified proteases had optimal activity at neutral to alkaline pH on azocasein, whereas their activity was optimal at acidic to neutral pH against 125I-insulin B-chain. The IC50 concentrations of 1,10-phenathroline against the two substrates, at the optimal pH, were comparable, suggesting that both activities measured were associated with gp63 rather than another contaminating enzyme. This was further supported by the comparable enrichment values, estimated from the specific activity of the enzyme during purification, using both assays. These results explain the earlier apparent discrepancies and suggest that the optimum pH of gp63 is substrate-dependent and not related to species differences or to the different purification procedures applied.
Tzinia
A K
AK
Laboratory of Biochemistry, Hellenic Pasteur Institute, Athens, Greece.
Soteriadou
K P
KP
D000818:Animals; D010450:Endopeptidases; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007891:Leishmania / Q000187:drug effects / Q000201:enzymology*; D007893:Leishmania donovani; D007894:Leishmania mexicana; D007895:Leishmania tropica; D008562:Membrane Glycoproteins / Q000302:isolation & purification* / Q000378:metabolism; D008666:Metalloendopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D015800:Protozoan Proteins / Q000302:isolation & purification* / Q000378:metabolism; D011817:Rabbits; D013379:Substrate Specificity / Q000187:drug effects
D008562:Membrane Glycoproteins; D015800:Protozoan Proteins; D010450:Endopeptidases; D008666:Metalloendopeptidases; C055146:glycoprotein gp63, Leishmania