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Article

Combined Interaction of Phospholipase C and Apolipoprotein A-I with Small Unilamellar Lecithin-Cholesterol Vesicles: Influence of Apolipoprotein A-I Concentration and Vesicle Composition^†

Manasa V. Gudheti,^‡ Sum P. Lee,^§ Dganit Danino, and Steven P. Wrenn*^‡

Department of Chemical and Biological Engineering, Drexel University, Philadelphia, Pennsylvania 19104, School of Medicine, University of Washington, Seattle, Washington 98195, and Department of Biotechnology and Food Engineering, Technion, Haifa, Israel 32000

Biochemistry, 2005, 44 (19), pp 7294–7304

DOI: 10.1021/bi047317m

Publication Date (Web): April 21, 2005

†

This work was supported in part by a Biomedical Engineering Research Grant from the Whitaker Foundation (RG-00-0417).

‡

Drexel University.

University of Washington.

Technion.

To whom correspondence should be addressed. Telephone: +1-215-895-6694. Fax: +1-215-895-5837. E-mail: wrenn@coe.drexel.edu.

Abstract

We report the combined effects of phospholipase C (PLC), a pronucleating factor, and apolipoprotein A-I (apo A-I), an antinucleating factor, in solutions of model bile. Results indicate that apo A-I inhibits cholesterol nucleation from unilamellar lecithin vesicles by two mechanisms. Initially, inhibition is achieved by apo A-I shielding of hydrophobic diacylglycerol (DAG) moieties so as to prevent vesicle aggregation. Protection via shielding is temporary. It is lost when the DAG/apo A-I molar ratio exceeds a critical value. Subsequently, apo A-I forms small (5−15 nm) complexes with lecithin and cholesterol that coexist with lipid-stabilized (400−800 nm) DAG oil droplets. This microstructural transition from vesicles to complexes avoids nucleation of cholesterol crystals and is a newly discovered mechanism by which apo A-I serves as an antinucleating agent in bile. The critical value at which a microstructural transition occurs depends on binding of apo A-I and so varies with the cholesterol mole fraction of vesicles. Aggregation of small, unilamellar, egg lecithin vesicles (SUVs) with varying cholesterol composition (0−60 mol %) was monitored for a range of apo A-I concentrations (2 to 89 μg/mL). Suppression of aggregation persists so long as the DAG-to-bound-apo A-I molar ratio is less than 100. A fluorescence assay involving dansylated lecithin shows that the suppression is an indirect effect of apo A-I rather than a direct inhibition of PLC enzyme activity. The DAG-to-total apo A-I molar ratio at which suppression is lost increases with cholesterol because of differences in apo A-I binding. Above this value, a microstructural transition to DAG droplets and lecithin/cholesterol A-I complexes occurs, as evidenced by sudden increases in turbidity and size and enhancement of Förster resonance energy transfer; structures are confirmed by cryo TEM.