Purification and characterization of an insect hemolymph lipoprotein ice nucleator: evidence for the importance of phosphatidylinositol and apolipoprotein in the ice nucleator activity

Summary A lipoprotein with ice nucleator activity was purified from the hemolymph of the freezetolerant larvae of the craneflyTipula trivittata. Characterization of this lipoprotein ice nucleator (LPIN) showed that it differed from other previously described insect hemolymph lipoproteins which lack ice nucleator activity, by the presence of phosphatidylinositol (PI) at 11.0% by weight of the total phospholipid content. The potential roles of PI and other lipoprotein components in the ice nucleating activity were examined using various phospholipases, proteases, LPIN antibodies, borate compounds and various lipid-protein reconstitutions. It was found that phosphatidylinositol specific phospholipase C was the most effective phospholipase in eliminating the activity of the LPIN. Borate compounds effectively depressed activity. Treatment of the LPIN with protease also eliminated ice nucleator activity but the binding of LPIN specific antibody did not. Reconstitutions consisting of the native LPIN lipids, PI specific phospholipase-treated native LPIN lipids, or pure standard phospholipids with the apolipoproteins of the LPIN andManduca sexta larval lipoproteins gave evidence that both the apolipoproteins of the LPIN and PI are necessary for the ice nucleating activity.Abbreviations LPIN polyclonal antibodies to lipoprotein ice nucleator - ANOVA analysis of variance - Apo-I apolipoprotein I - Apo-II apolipoprotein II - LPIN lipoprotein ice nucleator - PAGE polyacrylamide gel electrophoresis - PAS Periodoacetate-Schiff's base - PC phosphatidylcholine - PE phosphatidylethanolamine - PI phosphatidylinositol - SCP supercooling point (ice nucleation temperature) - SDS sodium dodecyl sulfate - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - TLC thin layer chromatography