A strategy for solubilizing delipidated apolipoprotein with lysophosphatidylcholine and reconstitution with phosphatidylcholine.
The apolipoproteins of insect lipophorin were dissociated in guanidinium chloride and isolated by gel permeation chromatography. Over 98% of the total lipid in lipophorin was associated with apolipophorin I (apoLp-I), thus suggesting this apolipoprotein to be the lipid binding component of the particle. ApoLp-I was delipidated with ethanol/ether and solubilized in buffer that contained radioactive lysophosphatidylcholine ([3H]LPC) above the critical micellar concentration. Sonic irradiation of radioactive phosphatidylcholine ([14C]PC) with [3H]LPC-solubilized apoLp-I at a molar ratio of 318 resulted in reconstituted lipophorin (RLp-I). [3H]LPC was bound to fatty acid free bovine serum albumin and was separated from RLp-I by density gradient ultracentrifugation and gel permeation chromatography. Negatively stained RLp-I particles were quasispherical with an average radium of 55 A, and their overall morphology and secondary structure were similar to those of native hemolymph lipophorin. The RLp-I particle had a rho = 1.137 g/mL, a Mr approximately 5.2 X 10(5), and a [14C]PC:apoLp-I molar ratio of 308. From the compositional analysis, molecular size, trypsinization, and lipolysis with phospholipase A2, we concluded that each RLp-I particle contained one molecule of apoLp-I and a monomolecular layer of [14C]PC. When injected into the hemolymph of adult moths in vivo, RLp-I was loaded with lipid, as judged by a decrease in its density both in the presence and in the absence of adipokinetic hormone. The similarities in morphology and immunology of RLp-I and native lipophorin, together with the ability of RLp-I to load lipid, suggest that reconstituted lipophorins may serve as models to probe lipophorin structure and function.
Kawooya
J K
JK
Department of Biochemistry, University of Arizona, Tucson 85721.
Wells
M A
MA
Law
J H
JH
D000818:Animals; D001053:Apolipoproteins / Q000276:immunology / Q000302:isolation & purification*; D002352:Carrier Proteins / Q000276:immunology / Q000302:isolation & purification; D002942:Circular Dichroism; D007120:Immunochemistry; D008055:Lipids / Q000302:isolation & purification; D008074:Lipoproteins; D008244:Lysophosphatidylcholines; D015394:Molecular Structure; D009036:Moths; D010713:Phosphatidylcholines; D012995:Solubility
D001053:Apolipoproteins; D002352:Carrier Proteins; D008055:Lipids; D008074:Lipoproteins; D008244:Lysophosphatidylcholines; D010713:Phosphatidylcholines; C032830:lipophorin