A preliminary approach to the separation of Leishmania cell-surface antigens.
The purpose of the current study was to characterize Leishmania cell-surface antigens by two different methods established for the purification of glycoproteins and proteins, and to point out a useful approach to define their size and mass heterogeneity. L. tropica parasites were initially isolated from patients with active cutaneous leishmaniasis and were then cultured in vitro. The parasite-cell layer was solubilised with 6 M guanidinium chloride (GuHCl) and subsequently prepared for the purification procedure. The methods used in this work were gel filtration chromatography and isopycnic density-gradient centrifugation. Because of the presence of a substantial amount of non-specific proteins in the culture medium, these methods were not effective alone in distinguishing these antigens. However, a good idea of their N-glycosylated structures could be obtained by using Periodic acid-Schiffs (PAS) and Con A lectin, and also size and mass heterogeneity. A combination of these methods effected a clear separation of the antigens. Amino acid analysis of the purified antigens was performed to positively identify them as well-known Leishmania cell-surface antigen gene products. The results confirmed the presence of more than one cell-surface antigen on the Leishmania parasite and the combination of gel chromatography and density-gradient centrifugation could be useful for their isolation.
Aksoy
Nurten
N
Department of Biochemistry, Faculty of Medicine, Atatürk University, Erzurum, Turkey. naksoy@harran.edu.tr
Ozbilge
Hatice
H
Keles
Salt
S
Iriadam
Mehmet
M
Vural
Hüjseyin
H
Akcay
Fatih
F
D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D000941:Antigens / Q000737:chemistry; D002462:Cell Membrane / Q000378:metabolism*; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D006023:Glycoproteins / Q000302:isolation & purification; D019791:Guanidine / Q000494:pharmacology; D006801:Humans; D007895:Leishmania tropica / Q000378:metabolism*; D012685:Sepharose / Q000494:pharmacology
D000596:Amino Acids; D000941:Antigens; D006023:Glycoproteins; D012685:Sepharose; D019791:Guanidine