A strategy for solubilizing delipidated apolipoprotein with lysophosphatidylcholine and reconstitution with phosphatidylcholine.

Abstract:

: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.

journal_name

Biochemistry

journal_title

Biochemistry

authors

Kawooya JK,Wells MA,Law JH

doi

10.1021/bi00442a019

subject

Has Abstract

pub_date

1989-08-08 00:00:00

pages

6658-67

issue

16

eissn

0006-2960

issn

1520-4995

journal_volume

28

pub_type

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