The long term goal of the project is to understand the regulation of lipoprotein lipase (LPL) at the levels of gene transcription, protein synthesis, secretion and degradation. The main hypothesis to be tested is that the interaction of LPL with cell surface heparan sulfate proteoglycans (HSPG's) is the major determinant in regulating the degradation of the newly synthesized lipase and therefore the net output of enzyme. The LPL-binding HSPG's will be purified by affinity chromatography on LPL-Affigel-10 columns as the major purification step. Antibodies to the holoproteins and the various core proteins will be prepared and the core proteins cloned by screening of lambda-gt11 libraries. The LPL-binding HSPG's cDNA will be expressed in Chinese Hamster ovary (CHO) cells. LPL cDNA will be transfected into wild type and mutant CHO cells with variable cell surface HSPG's. Turnover of LPL in these cells will be determined quantitatively (synthesis, secretion, degradation in terms of ng/hr/cell DNA). The coordinate regulation of LPL and HSPG's turnover by insulin, thyroxine and CAMP in cultured adipocytes will be determined. The HSPG's binding domains of LPL will be determined by the following strategy: First the lysyl residues which are not accessible in the LPL-HSPG complex will be defined with the modifying reagent pyridoxal-5'-phosphate; second, a library of 10 monoclonal antibodies will be screened for their ability to inhibit LPL binding to the adipocyte cell surface; third, epitopes of inhibiting antibodies will be determined; fourth, site directed mutagenesis in limited sequences of LPL will be conducted.
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