HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Komaromy, M. C. Articles by Reed, M. Search for Related Content PubMed PubMed Citation Articles by Komaromy, M. C. Articles by Reed, M. Social Bookmarking                      What's this?

Journal of Lipid Research, Vol 32, 963-975, Copyright © 1991 by Lipid Research, Inc.

ARTICLES

Expression of rat hepatic lipase in heterologous systems: evidence for different sites for interface binding and catalysis

MC Komaromy and M Reed
Research Institute, Palo Alto Medical Foundation, CA 94301.

Rat hepatic triglyceride lipase was expressed as a bacterial fusion protein and as a secreted protein in eukaryotic cells. The bacterial fusion construct coded for seven amino acids at the N-terminus which are not present in the hepatic lipase cDNA, but otherwise consisted of only the complete mature lipase sequence. Fusion protein was isolated as an insoluble product which did not have lipase or phospholipase activities; it was, however, active as an esterase when solubilized after preparative gel electrophoresis. The fusion protein was used to raise polyclonal antibodies that recognize native rat hepatic lipase and inhibit its activity. For eukaryotic expression, a full-length rat hepatic lipase cDNA clone was inserted into the metallothionein promoter expression vector pMTSV40polyA.Bam. Transfected CHO cells, induced with ZnSO4, secreted an immunoreactive protein of Mr approximately 57,000. A lipase-producing clonal cell line was isolated and used to characterize the enzyme. The protein was purified from serum-free medium by heparin-Sepharose and DEAE-Trisacryl M column chromatography. It was apparently identical to native rat hepatic lipase, with the exception of the conformation of the linkage of the sialic acids which form part of the N-linked carbohydrate complexes. The bacterial fusion protein, the CHO-produced lipase, and native rat hepatic lipase were all inhibited by phenylmethylsulfonyl fluoride, implying that they function catalytically as serine esterases. Substrate competition studies indicated that the esterase and lipase activities use the same active site; thus, the major defect in the fusion protein was probably in triglyceride substrate binding. These results suggest that interface binding and catalysis occur at different sites in the protein.
CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				S.-J. Lee, S. Kadambi, C. David, A. D. Cooper, and S. Y. Choi Association of hepatic lipase with proteoglycans stimulates the production of proteoglycans in vivo and in vitro J. Lipid Res., July 1, 2004; 45(7): 1266 - 1271. [Abstract] [Full Text] [PDF]

				C. Donner, S. Choi, M. Komaromy, and A. D. Cooper Accelerated lipoprotein uptake by transplantable hepatomas that express hepatic lipase J. Lipid Res., September 1, 1998; 39(9): 1805 - 1815. [Abstract] [Full Text]

				S. Y. Choi, I. J. Goldberg, L. K. Curtiss, and A. D. Cooper Interaction between ApoB and Hepatic Lipase Mediates the Uptake of ApoB-containing Lipoproteins J. Biol. Chem., August 7, 1998; 273(32): 20456 - 20462. [Abstract] [Full Text] [PDF]

				M. Komaromy, S. Azhar, and A. D. Cooper Chinese Hamster Ovary Cells Expressing a Cell Surface-anchored Form of Hepatic Lipase. CHARACTERIZATION OF LOW DENSITY LIPOPROTEIN AND CHYLOMICRON REMNANT UPTAKE AND SELECTIVE UPTAKE OF HIGH DENSITY LIPOPROTEIN-CHOLESTERYL ESTER J. Biol. Chem., July 12, 1996; 271(28): 16906 - 16914. [Abstract] [Full Text] [PDF]

				M. Z. Kounnas, D. A. Chappell, H. Wong, W. S. Argraves, and D. K. Strickland The Cellular Internalization and Degradation of Hepatic Lipase Is Mediated by Low Density Lipoprotein Receptor-related Protein and Requires Cell Surface Proteoglycans J. Biol. Chem., April 21, 1995; 270(16): 9307 - 9312. [Abstract] [Full Text] [PDF]