A strategy for solubilizing delipidated apolipoprotein with lysophosphatidylcholine and reconstitution with phosphatidylcholine
- Univ. of Arizona, Tucson (USA)
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 (({sup 3}H)LPC) above the critical micellar concentration. Sonic irradiation of radioactive phosphatidylcholine (({sup 14}C)PC) with ({sup 3}H)LPC-solubilized apoLp-I at a molar ratio of 318 resulted in reconstituted lipophorin I (RLp-I). ({sup 3}H)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 radius of 55{angstrom}, 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 M{sub r} {approx} 5.2 x 10{sup 5}, and a ({sup 14}C)PC:apoLp-I molar ratio of 308. From the compositional analysis, molecular size, trypsinization, and lipolysis with phospholipase A{sub 2}, the authors concluded that each RLp-I particle contained one molecule of apoLp-I and a monomolecular layer of ({sup 14}C)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.
- OSTI ID:
- 5353270
- Journal Information:
- Biochemistry; (USA), Journal Name: Biochemistry; (USA) Vol. 28:16; ISSN 0006-2960; ISSN BICHA
- Country of Publication:
- United States
- Language:
- English
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59 BASIC BIOLOGICAL SCIENCES
APOLIPOPROTEINS
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CARBON 14 COMPOUNDS
CHALCOGENIDES
DAYS LIVING RADIOISOTOPES
ELECTROPHORESIS
ESTERS
EVEN-ODD NUCLEI
HYDROGEN COMPOUNDS
ISOTOPES
LABELLED COMPOUNDS
LIGHT NUCLEI
LIPIDS
LIPOPROTEINS
MEMBRANES
MOLECULAR STRUCTURE
NUCLEI
ORGANIC COMPOUNDS
ORGANIC PHOSPHORUS COMPOUNDS
OXIDES
OXYGEN COMPOUNDS
PHOSPHOLIPIDS
PROTEINS
RADIOISOTOPES
SOLUBILITY
SULFUR 35
SULFUR ISOTOPES
TRITIUM COMPOUNDS
TRITIUM OXIDES