The objective of this project is to elucidate the mechanisms whereby the protein and lipid components of the high density lipoproteins are assembled in the liver and intestinal cells to form discoidal, nascent HDL and to investigate the metabolic transformation of these particles afte their secretion into the plasma compartment. In exploring the biogenesis of nascent HDL, we propose to test the hypothesis that subcellular membranes of low cholesterol content, particularly the endoplasmic reticulum, are preferentially susceptible to interaction with newly synthesized apolipoproteins, and thereby provide the lipid component of the nascent HDL. Products generated by the in vitro incubation of radiolabelled apoproteins and purified membrane fractions will be characterized with respect to their chemical and physical properties. To study the details of post-secretory processing of the nascent HDL, well-defined discoidal recombinants of apo A-I and apo E will be studied as models for the nascent particles. Recombinants of various sizes will be studied as substrates for lecithin: cholesterol acyl transferase (LCAT) to investigate the effects of particle size on this reaction. Apo A-IV and apo E from rat HDL will be studied as effector molecules for this enzyme. Changes in protein and lipid composition of the discoidal substrates resulting from LCAT action will also be investigated. Chemical cross-linking will be employed to study the existence and properties of the proposed plasma complex of LCAT, A-I, and a cholesteryl ester transfer protein. Study of the nature of the interaction between LCAT and apo A-I in such a complex will be initiated by performing chemical modifications of specific amino acid side chains. A comparison of effects on lipid-binding and activation of LCAT is expected to define the nature and importance of protein-protein interactions in the activation of LCAT by apo A-I.
