Cycloheximide sensitivity in regulation of acyl coenzyme A:cholesterol acyltransferase activity in Chinese hamster ovary cells. 1. Effect of exogenous sterols.
Chinese hamster ovary cells grown in medium containing low-density lipoprotein (LDL) express high acyl coenzyme A:cholesterol acyltransferase (ACAT) activity as measured by an [3H]oleate pulse. Removal of LDL from the medium causes rapid inactivation of ACAT activity; the t1/2 for the initial inactivation rate is 0.8 h. Preincubation with protein synthesis inhibitors (cycloheximide or emetine) for 2 h or longer lengthens the t1/2 for the initial inactivation rate to approximately 2.1 h. When LDL is removed for more than 10 h, the cells contain only 3% of the original ACAT activity. Cycloheximide under this condition causes an 8-fold increase in ACAT activity; the increase approaches a maximum in 6-8 h. The extent of ACAT activation by cycloheximide inversely depends on exogenous sterol present in the medium; LDL diminishes the activation, while cationized LDL or 25-hydroxycholesterol completely abolishes the activation. Adding LDL back to the sterol-free medium causes a 40-70-fold increase in ACAT activity; however, the activation of LDL is not further augmented if the cells are pretreated with cycloheximide. The above observations are qualitatively confirmed by ACAT assays in vitro with cell homogenates. LDL or cycloheximide has no effect on the rates of 3H-labeled triglyceride and 3H-labeled polar lipid synthesis. Efflux of prelabeled cholesterol from cells is cycloheximide-insensitive. Rates of degradation of [3H]-leucine-pulse-labeled total protein in cells grown with or without LDL are identical. The above results imply the existence of at least one specific short-lived factor that directly or indirectly inhibits ACAT activity.(ABSTRACT TRUNCATED AT 250 WORDS)
C C
CC
Chang
G M
GM
Doolittle
T Y
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Chang
D000818:Animals; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D002784:Cholesterol / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D003470:Culture Media; D003513:Cycloheximide / Q000494:pharmacology*; D004789:Enzyme Activation; D005260:Female; D006801:Humans; D006888:Hydroxycholesterols / Q000494:pharmacology*; D007700:Kinetics; D008077:Lipoproteins, LDL / Q000494:pharmacology; D010053:Ovary; D002785:Sterol O-Acyltransferase / Q000378:metabolism*
D003470:Culture Media; D006888:Hydroxycholesterols; D008077:Lipoproteins, LDL; C007997:25-hydroxycholesterol; D002784:Cholesterol; D003513:Cycloheximide; D002785:Sterol O-Acyltransferase