Purification and characterization of a leucine aminopeptidase from the bovine filarial parasite Setaria cervi.
Using synthetic peptide substrate Leu-p-NA, leucine aminopeptidase (LAP) activity was detected in both microfilarial and adult stages of a bovine filarial parasite Setaria cervi. A single protein fraction containing LAP activity was purified from the adult female S. cervi using three different chromatographic techniques. This purified enzyme was shown to be a 321 kDa zinc dependent metalloexopeptidase having maximum activity at pH 9.0 and 37 degrees C. Its activity was significantly inhibited by aminopeptidase specific inhibitors such as 1,10-phenanthroline, ethylene diaminetetraacetic acid (EDTA), amastatin and bestatin; and activated by Co2+, Mn2+ and Mg2+ ions. Puromycin and l-amino acids (e.g., glutamine, leucine and glycine) also showed some moderate inhibitory effects on the purified enzyme. Among various synthetic substrates tested, the purified enzyme hydrolysed Leu-p-NA at very high rate suggesting it to be a LAP. Both ELISA and western blotting analyses of S. cervi LAP revealed the presence of homologous protein in human filarial parasite Wuchereria bancrofti. The higher sensitivity of S. cervi LAP with microfilariaemic sera compared to other categories of W. bancrofti infected human sera implied its potential as a serodiagnostic marker against active filarial infection. The antigenic similarity between S. cervi LAP and W. bancrofti makes this molecule ideal for the discovery of new diagnostic marker, drugs and/or vaccine candidate for human lymphatic filariasis.
Pokharel
Daya Ram
DR
Department of Biochemistry, Faculty of Sciences, Banaras Hindu University, Varanasi 221005, UP, India.
Rathaur
Sushma
S
D000818:Animals; D000909:Antibodies, Helminth / Q000097:blood; D015153:Blotting, Western; D002845:Chromatography; D020536:Enzyme Activators / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005370:Filarioidea / Q000201:enzymology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007931:Leucyl Aminopeptidase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification*; D008970:Molecular Weight; D013379:Substrate Specificity; D013696:Temperature; D014958:Wuchereria bancrofti / Q000201:enzymology; D015032:Zinc / Q000494:pharmacology
D000909:Antibodies, Helminth; D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D007931:Leucyl Aminopeptidase; D015032:Zinc