The GOALS of the proposed research are: To determine how the major constituents (apoproteins, triacylglycerol, cholesterol, and phospholipids) of very low density lipoprotein are assembled and translocated and to determine how these processes and subsequent secretion are regulated. To determine the mechanism(s) by which acetyl-CoA carboxylase--the key regulatory enzyme of fatty synthesis, hence de novo lipogenesis--is controlled. The estrogen-induced chick hepatocyte in monolayer culture, a system developed in this laboratory, will serve as experimental model. The chick hepatocyte is ideally suited for studies on VLDL synthesis and secretion, since the liver accounts for nearly all de novo fatty acid synthesis in the chick, as it dies in man, and provides the major fraction of the fatty acids utilized by other tissues (including adipose tissue) in the form of VLDL -triacylglycerol, and since VLDL output of the hepatocyte in culture can be increased 10-20-fold by prior hormone treatment. We have already amassed a large body of background information with this system. The sequence of events will be determined by which apoproteins and lipids are assembled to form the nascent VLDL particle. The intracellular sites of these events will be identified and characterized. Using puromycin-induced nascent chain release, the interaction of segments of the apo-B polypeptide and specific VLDL lipids will be established. A cell-free system will be developed that is capable of assembling, processing and translocating nascent VLDL. This cell-free system will be used to investigate the minimal requirements of and the mechanisms of key steps in the VLDL assembly/processing pathway. cDNA clones which encode the mRNA for apo-B will be isolated and sequenced. The regulatory mechanisms for the synthesis, assembly, translocation and secretion of VLDL will be investigated. The effect of the phosphorylation state of acetyl-CoA carboxylase on the kinetics of its citrate-induced activation and polymerization and the state of its protomerpolymer equilibrium will be determined.