Cell-free biosynthesis of lipophosphoglycan from Leishmania donovani. Characterization of microsomal galactosyltransferase and mannosyltransferase activities.
Incubation of microsomal preparations from Leishmania donovani parasites with UDP-[3H]galactose or GDP-[14C]mannose resulted in incorporation of radiolabel into an endogenous product that exhibited the chemical and chromatographic characteristics of the parasite's major surface glycoconjugate, lipophosphoglycan. The [3H]galactose- or [14C]mannose-labeled product was (i) cleaved by phosphatidylinositol-specific phospholipase C; (ii) deaminated by nitrous acid; and (iii) degraded into radioactive, low molecular weight fragments upon hydrolysis with mild acid. Analysis of the products of mild acid hydrolysis revealed the presence of phosphorylated Gal-beta-Man as the major fragment with lesser amounts of mono-, tri-, and tetrasaccharides. The incorporation of the two isotopic precursors was neither stimulated by the addition of dolichylphosphate nor inhibited by amphomycin, indicating that dolichol-saccharide intermediates are not involved in assembly of the repeating units of lipophosphoglycan. Development of this cell-free glycosylating system will facilitate further studies on the pathway and enzymes involved in lipophosphoglycan biosynthesis.
Carver
M A
MA
Department of Biochemistry, University of Kentucky Medical Center, Lexington 40536.
Turco
S J
SJ
D000818:Animals; D002250:Carbon Radioisotopes; D002474:Cell-Free System; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002854:Chromatography, Paper; D005690:Galactose / Q000378:metabolism; D005700:Galactosyltransferases / Q000378:metabolism*; D006028:Glycosphingolipids / Q000096:biosynthesis* / Q000302:isolation & purification; D007425:Intracellular Membranes / Q000378:metabolism; D007700:Kinetics; D007893:Leishmania donovani / Q000201:enzymology*; D008358:Mannose / Q000378:metabolism; D008364:Mannosyltransferases / Q000378:metabolism*; D008861:Microsomes / Q000201:enzymology*; D009844:Oligosaccharides / Q000096:biosynthesis / Q000302:isolation & purification; D014316:Tritium
D002250:Carbon Radioisotopes; D006028:Glycosphingolipids; D009844:Oligosaccharides; C008290:lipophosphonoglycan; D014316:Tritium; D005700:Galactosyltransferases; D008364:Mannosyltransferases; D008358:Mannose; D005690:Galactose