Human lipoprotein lipase (LPL) and hepatic lipase (HL) will be sequenced and structural domains on these enzymes identified. Several strategies will be employed. cDNA's for human HL and LPL mRNA will be isolated by screening, with reduced stringency, human genomic libraries with rat and chicken cDNA's and exploiting the expected sequence homologies. Proteolytic and cyanogen bromide fragments will be separated by reversed phase chromatography and sequenced. Hybridoma producing monoclonal antibodies (mAb) to lipolytic enzymes will be generated by improved methods which will enhance the selection of mAb's to exposed epitopes of the native HL and LPL. Monoclonal antibodies will be assigned to specific proteolytic and cyanogen bromide fragments and to peptides generated in vitro in expression vectors. Characterized mAb's will be evaluated for their ability to inhibit functional properties of lipases: Binding of enzyme to substrate and heparan sulfate; catalytic activity; binding of Apo CII to LPL; dimer formation from monomeric LPL. The function of HL will be investigated by testing the following hypothesis: HL is a major regulator of chylomicron and VLDL removal by unmasking specific domains on Apo E which are necessary for recognition by the chylomicron receptor. Chylomicron remnants will be isolated from HL-deficient rats and their binding characteristic to liver membranes measured after gradual digestion with HL. Unmasking of Apo E domains by HL will be monitored by reactivity with monoclonal antibodies, ligand blotting of separated liver membrane proteins and in vivo removal rate of modified lipoproteins. Finally HL regulation in rat hepatocytes by hormones (insulin, glucagon, thyroxine) will be studied at the level of synthesis, degradation, secretion, HL mRNA concentration and transcription. Hormone responsive sequences in the 5'-flanking region of the HL gene will be identified.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Metabolism Study Section (MET)
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Cornell University
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Montalto, M B; Bensadoun, A (1993) Lipoprotein lipase synthesis and secretion: effects of concentration and type of fatty acids in adipocyte cell culture. J Lipid Res 34:397-407
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Strieleman, P J; Bensadoun, A (1987) Cell-free translation of avian adipose tissue lipoprotein lipase messenger RNA. Biochim Biophys Acta 908:268-74
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