Serine protease activities in Leishmania (Leishmania) chagasi promastigotes.
The present work reports the isolation, biochemical characterization, and subcellular location of serine proteases from aqueous, detergent soluble, and culture supernatant of Leishmania chagasi promastigote extracts, respectively, LCSII, LCSI, and LCSIII. The active enzyme molecular masses of LCSII were about 105, 66, and 60 kDa; of LCSI, 60 and 58 kDa; and of LCSIII, approximately 76 and 68 kDa. Optimal pH for the enzymes was 7.0 for LCSI and LCSIII and 8.5 for LCSII, and the optimal temperature for all enzymes was 37°C, using α-N-ρ-tosyl-L: -arginine methyl ester as substrate. Assay of thermal stability indicated that LCSIII is the more stable enzyme. Hemoglobin, bovine serum albumin, and ovalbumin were hydrolyzed by LCSII and LCSI but not by LCSIII. Inhibition studies suggested that enzymes belong to the serine protease class modulated by divalent cations. Rabbit antiserum against 56-kDa serine protease of Leishmania amazonensis identified proteins in all extracts of L. chagasi. Furthermore, immunocytochemistry demonstrated that serine proteases are located in flagellar pocket region and cytoplasmic vesicles of L. chagasi promastigotes. These findings indicate that L. chagasi serine proteases differ from L. amazonensis proteases and all known flagellate proteases, but display some similarities with serine proteases from other Leishmania species, suggesting a conservation of this enzymatic activity in the genus.
da Silva-López
Raquel Elisa
RE
Laboratório de Bioquímica de Proteínas e Peptídeos, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, Rio de Janeiro, Brazil. rlopez@far.fiocruz.br
dos Santos
Tatiana Resende
TR
Morgado-Díaz
José Andrés
JA
Tanaka
Marcelo Neves
MN
de Simone
Salvatore Giovanni
SG
D000818:Animals; D000913:Antibodies, Protozoan / Q000276:immunology; D002413:Cations, Divalent / Q000378:metabolism; D003067:Coenzymes / Q000378:metabolism; D004795:Enzyme Stability; D006454:Hemoglobins / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007891:Leishmania / Q000201:enzymology*; D008856:Microscopy, Fluorescence; D008970:Molecular Weight; D015388:Organelles / Q000201:enzymology; D010047:Ovalbumin / Q000378:metabolism; D015800:Protozoan Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011817:Rabbits; D057057:Serine Proteases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D012710:Serum Albumin, Bovine / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature
D000913:Antibodies, Protozoan; D002413:Cations, Divalent; D003067:Coenzymes; D006454:Hemoglobins; D015800:Protozoan Proteins; D012710:Serum Albumin, Bovine; D010047:Ovalbumin; D057057:Serine Proteases