EARLY STEPS IN GLYCOSYLPHOSPHATIDYLINOSITOL BIOSYNTHESIS IN LEISHMANIA-MAJOR

A cell-free system based on washed Leishmania major membranes was labe lled with GDP-[H-3]Man in the presence of synthetic glucosaminyl-phosp hatidylinositol (GlcN-PI) and N-acetylglucosaminyl-phosphatidylinosito l (GlcNAc-PI). In both cases, the major radiolabelled products were Ma n alpha 1-4GlcN alpha 1 -6myo-inositoll-HPO4-(sn-1,2-dipalmitoylglycer ol) and Man alpha 1-4GlcN alpha 1-6myo-inositoll-HPO4- (sn-1-palmitoyl -2-lyso-glycerol), to which an additional D-mannose residue was added when a chase with an excess of GDP-Man was performed. The L. major cel l-free system can therefore be used to observe the actions of four enz ymes, namely GlcNAc-PI de-N-acetylase, Dol-P-Man-GlcN-PI alpha 1-4-man nosyltransferase, a phospholipase A(2)-like activity and a second alph a-mannosyltransferase activity. The substrate specificities of the fir st two of these enzymes were studied using a series of substrate analo gues. GlcNAc-PI de-N-acetylase was tested against a variety of N-acyla ted GlcN-PI substrates and was able to cleave N-acetyl and N-propyl gr oups but not larger groups such as N-butyl, N-isobutyl, N-pentyl and N -hexyl. The Dol-P-Man-GlcN-PI alpha 1-4-mannosyltransferase activity r equired the amino group of the glucosamine residue and the D-configura tion of the myo-inositol residue of the GlcN-PI acceptor substrate.