title: Biotinylation of lipoprotein lipase and hepatic triglyceride lipase: application in the assessment of cell binding sites.
abstract: Lipoprotein lipase (LPL) and hepatic triglyceride lipase (HL) were biotinylated using N-hydroxysuccinamide ester of biotin (25-fold molar excess) which was incorporated into the lysine amino groups of the enzyme protein. By assessing enzyme activity and heparin-agarose affinity a biotinylation protocol which did not denature lipases was developed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that biotinylated LPL (bLPL) has the same mobility as that of unlabeled or iodinated LPL. Receptor binding activity of bLPL was studied in (i) cell binding experiments using cultured bovine aortic endothelial cells and (ii) ligand blotting experiments using endothelial cell plasma membranes. Endothelial cells in culture bound similar amounts of bLPL and 125I-LPL. We previously described a 116-kDa heparin-releasable LPL binding protein (hrp-116) on endothelial cells. Using biotinylated lipases in ligand blotting experiments we now demonstrate that both bLPL and biotinylated HL can bind to hrp-116. bLPL in addition also bound to low-density lipoprotein receptor related protein in ligand blotting. Thus, our protocol has produced biotinylated lipases which are both chemically and biologically active and can be used instead of iodinated lipases.
authors.affiliation: Department of Medicine, Columbia University, New York, New York 10032.
authors.forename: P
authors.initials: P
authors.lastname: Sivaram
authors.forename: S
authors.initials: S
authors.lastname: Wadhwani
authors.forename: M G
authors.initials: MG
authors.lastname: Klein
authors.forename: A
authors.initials: A
authors.lastname: Sasaki
authors.forename: I J
authors.initials: IJ
authors.lastname: Goldberg
mesh_terms: D000818:Animals; D001710:Biotin; D002417:Cattle; D002478:Cells, Cultured; D002847:Chromatography, Agarose; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism; D015151:Immunoblotting; D008024:Ligands; D008049:Lipase / Q000737:chemistry*; D008071:Lipoprotein Lipase / Q000737:chemistry*; D008099:Liver / Q000201:enzymology*; D011956:Receptors, Cell Surface / Q000032:analysis*
chemical_list: D008024:Ligands; D011956:Receptors, Cell Surface; D001710:Biotin; D008049:Lipase; D008071:Lipoprotein Lipase