The objective of this proposal is to investigate the mechanisms of regulation of lipoprotein lipase (LPL) biosynthesis, processing, secretion and catalytic efficiency by hormones and other metabolic regulators. On the basis of results on hand the following hypotheses are proposed and will be evaluated. (1) Dibutyryl cAMP and lipolytic hormones (glucagon) decrease the amount of cellular LPL protein and the rate of secretion of the enzyme by increasing its intracellular degradation rate. Phosphorylation of the enzyme does not affect its catalytic efficiency, but enhances its degradation rate. Insulin opposes this effect and increases cellular LPL and its secretion by inducing an increase in cytoplasmic LPL mRNA. (2) Heparin and other glycosaminoglycan molecules with high affinity for LPL increase the rate of synthesis and secretion of this enzyme by decreasing the occupancy of LPL binding sites and increasing binding site numbers on the plasma membrane and intracellular membranes. (3) LPL is synthesized as a pre-LPL pre-cursor which is contranslationally glycosylated. Glycosylation of the enzyme may affect catalytic activity and is crucial for proper processing and secretion of LPL. In these studies relative rates of synthesis of adipocyte LPL in culture will be determined by incorporation of [S35] methionine into enzyme protein over 15 to 20 min. Degradation rates will be determined in pulse chase experiments, where cells are incubated for 60 min in the presence of [S35] methionine before addition of excess medium containing unlabeled methionine. Phosphorylation of LPL in whole adipocytes and in cell free systems will be followed by incorporation of [P32] phosphate or Gamma[P32]ATP into enzyme protein. Should the enzyme be phosphorylated, proteolytic fragments phosphorylated will be identified in LPL isolated from cells treated with dibutyryl cAMP and or insulin. Enzyme protein will be isolated by quantitative and specific immunoadsorption on affinity purified anti-LPL immunoglobulins coupled to polyacrylamide beads. Enzyme protein will be determined by a noncompetitive solid phase RIA or by a noncompetitive ELISA assay utilizing a monoclonal antibody to LPL. The specificity of the stimulation of LPL synthesis by heparin will be quantitatively evaluated by comparing the potency of crude heparin with heparin subfractions purified on LPL-Sepharose columns, or subfractions of heparan sulfate isolated from cultured endothelial cells or smooth muscle cells. Specific LPL mRNA activity will be determined by in vitro cell free translation and mRNA cellular content by hybridization with an LPL cDNA probe. The LPL gene will be cloned using the lambda gtll expression vector.
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