Effect of carbonyl cyanide m-chlorophenylhydrazone (CCCP) on the dimerization of lipoprotein lipase.
Lipoprotein lipase (LPL), an enzyme playing the central role in triglyceride metabolism, is a glycoprotein and a homodimer of identical subunits. Dimerization and proper processing of oligosaccharide chains are important maturation steps in post-translational regulation of enzyme activity. Indirect evidences suggest that dimerization of LPL occurs in endoplasmic reticulum (ER) or Golgi. In this study, we investigated the dimerization status of LPL in 3T3-L1 adipocytes, using sucrose density gradient ultracentrifugation and carbonyl cyanide m-chlorophenylhydrazone (CCCP), an inhibitor of ER-Golgi protein transport. In the presence of CCCP, no increase of cellular LPL activity was detected during 2 b of recovery period after the depletion of LPL, with heparin and cycloheximide. Only endoglycosidase H (endo H)-sensitive subunits were found in CCCP-treated cells after endo H digestion, suggesting that inactive LPL was retained in ER. In the presence of castanospermine, an inhibitor of ER glucosidase I, LPL subunits of both control and CCCP-treated cells had same molecular weight, indicating that complete oligosaccharides were transferred to LPL subunits in the presence of CCCP. In sucrose density gradient ultracentrifugation, all the LPL protein synthesized in the presence of CCCP was found at the dimeric fractions as in control cells. Most of LPL protein in control cells showed high affinity for heparin, and there was no difference between the control and CCCP-treated cells. These results suggest that dimerization and acquisition of high affinity for heparin of LPL can occur in ER of CCCP-treated cells without acquisition of catalytic activity.
Park
J W
JW
Department of Biochemistry, Chonbuk National University, Medical School, Chonju, South Korea. jinwoo@moak.chonbuk.ac.kr
Lee
S Y
SY
Yang
J Y
JY
Rho
H W
HW
Park
B H
BH
Lim
S N
SN
Kim
J S
JS
Kim
H R
HR
D016475:3T3 Cells; D000273:Adipose Tissue / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D000818:Animals; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology*; D019281:Dimerization; D004721:Endoplasmic Reticulum / Q000201:enzymology; D006031:Glycosylation; D006056:Golgi Apparatus / Q000201:enzymology; D006493:Heparin / Q000378:metabolism; D008071:Lipoprotein Lipase / Q000737:chemistry* / Q000378:metabolism*; D051379:Mice; D008970:Molecular Weight; D014475:Uncoupling Agents / Q000494:pharmacology*
D014475:Uncoupling Agents; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D006493:Heparin; D008071:Lipoprotein Lipase