J. Lipid Res. Acyl Labeled PIP's available August 1, 2008
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chisholm, J. W.
Right arrow Articles by Parks, J. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chisholm, J. W.
Right arrow Articles by Parks, J. S.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?
The Journal of Lipid Research, Vol. 40, 1512-1519, August 1999
Copyright © 1999 by Lipid Research, Inc.

Original Article

Characterization of C-terminal histidine-tagged human recombinant lecithin:cholesterol acyltransferase

Jeffrey W. Chisholma, Abraham K. Gebrea, and John S. Parksa
a Department of Pathology, Section on Comparative Medicine, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157 -1040

Correspondence to: John S. Parks

Lecithin:cholesterol acyltransferase (LCAT) is the plasma enzyme that catalyzes esterification of the sn-2 fatty acid of phospholipid to cholesterol. To facilitate the isolation of large quantities of LCAT and to assist in future structure;–function studies, LCAT containing a carboxy-terminal histidine-tag (H6) was expressed in Chinese hamster ovary cells (CHO). A high level of CHO-hLCATH6 expression ({approx}15 mg L-1) was achieved over a 72-h period using 10 mM sodium butyrate to enhance transcription and PFX-CHO protein-free medium. The pure enzyme ({approx}96%) was isolated by cobalt metal affinity chromatography with an activity yield of 82 ± 26%. CHO-hLCATH6 and CHO-hLCAT species had identical specific activities (26 ± 6 and 26 ± 3 nmol CE formed µg-1 h-1, respectively). The enzymatic activity of CHO-hLCATH6 was stable at 4°C in excess of 60 days. Substrate saturation studies, using rHDL composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), cholesterol, and apolipoprotein A-I (80:5:1) indicated that the appKm for CHO-hLCATH6, CHO-hLCAT, and purified plasma LCAT were nearly identical at {approx}2 µM substrate cholesterol.

We conclude that carboxy-terminal histidine-tagged LCAT is a suitable replacement for both plasma LCAT and CHO-hLCAT.—Chisholm, J. W., A. K. Gebre, and J. S. Parks. Characterization of C-terminal histidine-tagged human recombinant lecithin:cholesterol acyltransferase. J. Lipid Res. 1999. 40: 1512;–1519.

Supplementary key words: phosphatidylcholine-O-cholesterol acyltransferase, LCAT, CHO cells, cobalt metal affinity chromatography, butyric acid, his-tag, glycosylation analysis, PFX-CHO


Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Lipid Res.Home page
J.-Y. Lee, R. M. Badeau, A. Mulya, E. Boudyguina, A. K. Gebre, T. L. Smith, and J. S. Parks
Functional LCAT deficiency in human apolipoprotein A-I transgenic, SR-BI knockout mice
J. Lipid Res., May 1, 2007; 48(5): 1052 - 1061.
[Abstract] [Full Text] [PDF]

Home page
 J. Lipid Res.Home page
Y. Zhao, A. K. Gebre, and J. S. Parks
Amino acids 149 and 294 of human lecithin:cholesterol acyltransferase affect fatty acyl specificity
J. Lipid Res., December 1, 2004; 45(12): 2310 - 2316.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
R. E. Temel, A. K. Gebre, J. S. Parks, and L. L. Rudel
Compared with Acyl-CoA:Cholesterol O-Acyltransferase (ACAT) 1 and Lecithin:Cholesterol Acyltransferase, ACAT2 Displays the Greatest Capacity to Differentiate Cholesterol from Sitosterol
J. Biol. Chem., November 28, 2003; 278(48): 47594 - 47601.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
R. E. Temel, J. S. Parks, and D. L. Williams
Enhancement of Scavenger Receptor Class B Type I-mediated Selective Cholesteryl Ester Uptake from apoA-I-/- High Density Lipoprotein (HDL) by Apolipoprotein A-I Requires HDL Reorganization by Lecithin Cholesterol Acyltransferase
J. Biol. Chem., February 7, 2003; 278(7): 4792 - 4799.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
H.-h. Li, D. S. Lyles, W. Pan, E. Alexander, M. J. Thomas, and M. G. Sorci-Thomas
ApoA-I Structure on Discs and Spheres. VARIABLE HELIX REGISTRY AND CONFORMATIONAL STATES
J. Biol. Chem., October 11, 2002; 277(42): 39093 - 39101.
[Abstract] [Full Text] [PDF]

Home page
 J. Lipid Res.Home page
H. Liu, C. Labeur, C.-F. Xu, R. Ferrell, L. Lins, R. Brasseur, M. Rosseneu, K. M. Weiss, S. E. Humphries, and P. J. Talmud
Characterization of the lipid-binding properties and lipoprotein lipase inhibition of a novel apolipoprotein C-III variant Ala23Thr
J. Lipid Res., November 1, 2000; 41(11): 1760 - 1771.
[Abstract] [Full Text]

Home page
 J. Biol. Chem.Home page
L. Krimbou, M. Marcil, J. Davignon, and J. Genest Jr.
Interaction of Lecithin:Cholesterol Acyltransferase (LCAT){middle dot}alpha 2-Macroglobulin Complex with Low Density Lipoprotein Receptor-related Protein (LRP). EVIDENCE FOR AN alpha 2-MACROGLOBULIN/LRP RECEPTOR-MEDIATED SYSTEM PARTICIPATING IN LCAT CLEARANCE
J. Biol. Chem., August 24, 2001; 276(35): 33241 - 33248.
[Abstract] [Full Text] [PDF]


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Journal of Biological Chemistry 
 Molecular and Cellular Proteomics   ASBMB Today 
Copyright © 1999 by the American Society for Biochemistry and Molecular Biology.