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.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL024873-12
Application #
3337856
Study Section
Metabolism Study Section (MET)
Project Start
1979-12-01
Project End
1993-03-31
Budget Start
1991-04-01
Budget End
1992-03-31
Support Year
12
Fiscal Year
1991
Total Cost
Indirect Cost
Name
Cornell University
Department
Type
Other Domestic Higher Education
DUNS #
City
Ithaca
State
NY
Country
United States
Zip Code
14850
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
Brasaemle, D L; Cornely-Moss, K; Bensadoun, A (1993) Hepatic lipase treatment of chylomicron remnants increases exposure of apolipoprotein E. J Lipid Res 34:455-65
Bensadoun, A (1991) Lipoprotein lipase. Annu Rev Nutr 11:217-37
Hoogewerf, A J; Cisar, L A; Evans, D C et al. (1991) Effect of chlorate on the sulfation of lipoprotein lipase and heparan sulfate proteoglycans. Sulfation of heparan sulfate proteoglycans affects lipoprotein lipase degradation. J Biol Chem 266:16564-71
Sensel, M G; Legrand-Lorans, A; Wang, M E et al. (1990) Isolation and characterization of clones for the rat hepatic lipase gene upstream regulatory region. Biochim Biophys Acta 1048:297-302
Cisar, L A; Hoogewerf, A J; Cupp, M et al. (1989) Secretion and degradation of lipoprotein lipase in cultured adipocytes. Binding of lipoprotein lipase to membrane heparan sulfate proteoglycans is necessary for degradation. J Biol Chem 264:1767-74
Cisar, L A; Bensadoun, A (1987) Characterization of the intracellular processing and secretion of hepatic lipase in FU5AH rat hepatoma cells. Biochim Biophys Acta 927:305-14
Strieleman, P J; Bensadoun, A (1987) Cell-free translation of avian adipose tissue lipoprotein lipase messenger RNA. Biochim Biophys Acta 908:268-74
Cupp, M; Bensadoun, A; Melford, K (1987) Heparin decreases the degradation rate of lipoprotein lipase in adipocytes. J Biol Chem 262:6383-8
Bensadoun, A; Marita, R A (1986) Dibutyryl cyclic AMP decreases the rate of lipoprotein lipase synthesis in cultured adipocytes. Biochim Biophys Acta 879:253-63

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