EFFECT OF PHOSPHOLIPASE-C AND APOLIPOPHORIN-III ON THE STRUCTURE AND STABILITY OF LIPOPHORIN SUBSPECIES

Four distinct subspecies of the insect hemolymph lipoprotein, lipophor in, that range in diacylglycerol (DAG) content from approximately 100 to 1000 molecules per particle, were treated with phospholipase C. Lip id analysis demonstrated that both phosphatidylcholine and phosphatidy lethanolamine were hydrolyzed to DAG. Phospholipase C was used to remo ve 74-82% of the phospholipid of different lipophorins and these were analyzed for aggregation. Low density lipophorin (LDLp), the largest s ubspecies, with a diameter of similar to 23 nm, developed turbidity (m onitored by sample absorbance at 340 nm) suggesting the formation of l ipoprotein aggregates. High density lipophorin-adult (HDLp-A) and high density lipophorin-wanderer 1 (HDLp-W1) also displayed an increase in A(340) when incubated with phospholipase C, although the maximal incr ease observed was considerbly less than that for LDLp on a per particl e basis. Phospholipase C caused only a minimal increase in A340 in a f ourth subspecies, high density lipophorin-wanderer 2 (HDLp-W2), which contains an even lower amount of DAG. Electron microscopy was used to evaluate changes in particle morphology as a result of phospholipid de pletion. HDLp-W2 and HDLp-W1 showed signs of progressive aggregation a nd particle fusion. A similar aggregation/fusion was seen in the case of high density lipophorin adult (HDLp-A) while LDLp samples contained multiple aggregation/fusion foci and resultant very large particles. In the presence of exogenous apolipophorin III (apoLp-III), phospholip ase C-induced lipophorin aggregation/fusion was prevented. Electron mi croscopy of LDLp and HDLp-A samples revealed that apoLp-III-stabilized , phospholipase C-treated particles had a morphology similar to that o f control particles. Sodium dodecyl sulfate polyacrylamide gel electro phoresis of HDLp-W1, HDLp-A, and LDLp after incubation with phospholip ase C and apoLp-III demonstrated the association of apoLp-III with the se lipoproteins. Scanning densitometry of the stained gels showed that phospholipase C-treated, apoLp-III-stabilized lipophorin samples acqu ired 3-5 apoLp-III molecules/ particle as a result of phospholipase C- catalyzed phospholipid conversion to DAG. Thus, these experiments esta blish a correlation between the generation of DAG and the binding of a poLp-III to lipophorin particles. Furthermore, they provide direct evi dence that association of apoLp-III with DAG-enriched lipophorins func tions to stabilize particle structure.