TRANSBILAYER INHIBITION OF PROTEIN-KINASE-C BY THE LIPOPHOSPHOGLYCAN FROM LEISHMANIA-DONOVANI

Lipophosphoglycan (LPG), the predominant molecule on the surface of th e parasite Leishmania donovani, has previously been shown to be a pote nt inhibitor of protein kinase C (PKC) isolated from rat brain. The me chanism by which LPG inhibits PKC was further investigated in this stu dy. LPG was found to inhibit the PKCalpha-catalyzed phosphorylation of histone in assays using large unilamellar vesicles composed of 1-palm itoyl, 2-oleoyl phosphatidylserine and 1-palmitoyl, 2-oleoyl phosphati dylcholine either with or without 1% 1,2 diolein added. The results al so indicated that while PKC binding to sucrose-loaded vesicles was not substantially reduced in the presence of LPG at concentrations of 1-2 %, the activity of membrane-bound PKC was inhibited by 70%. This inhib ition of the membrane-bound form of PKC is not a consequence of reduce d substrate availability to the membrane. However, K-m shifted from ap proximate to 31 +/- 4 mu M to 105 +/- 26 mu M in the presence of 5% LP G. LPG caused PKC to bind to membranes without inducing a conformation al change as revealed by the lack of an increased susceptibility to tr ypsin. An LPG fragment containing only one repeating disaccharide unit was not as effective as the entire LPG molecule or of larger fragment s in inhibiting the membrane-bound form of the enzyme. The shorter fra gments were also less potent in raising the bilayer to hexagonal phase transition temperature of a model membrane. LPG is also able to inhib it the membrane-bound form of PKCalpha from the inner monolayer of lar ge unilamellar vesicles, the opposite monolayer to which the enzyme bi nds in our assay. Inhibition is likely a result of alterations in the physical properties of the membrane. To our knowledge, this is the fir st example of a membrane additive that can inhibit the membrane-bound form of PKC in the presence of other lipid cofactors.