Phosphorylation of native 97-kDa 3-hydroxy-3-methylglutaryl-coenzyme A reductase from rat liver. Impact on activity and degradation of the enzyme

R. A. Parker, Steven Miller, D. M. Gibson

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Abstract

Immunoprecipitation of native rat liver microsomal 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, phosphorylated by [γ-32P]ATP in the presence of reductase kinase, revealed a major 97-kDa 32P band which disappeared upon competition with pure unlabeled 53-kDa HMG-CoA reductase. A linear correlation between the expressed/total HMG-CoA reductase activity ratio (E/T) and the fraction of 32P released from the 97-kDa enzyme established the validity of the E/T ratio as an index of HMG-CoA reductase phosphorylation state in isolated microsomes. Incubation of rat hepatocytes with mevalonolactone resulted in a rapid increase in phosphorylation of microsomal reductase (decrease in E/T) followed by an enhanced rate of decay of total reductase activity which was proportional to the loss of 97-kDa enzyme mass determined by immunoblots. Inhibitors of lysosome function dampened both basal and mevalonate-induced reductase degradation in hepatocytes. In an in vitro system using the calcium-dependent protease calpain-2, up to 5-fold greater yields of soluble 52-56-kDa fragments of reductase (immunoblot and total activity) were obtained when the substrate 97-kDa reductase was phosphorylated before proteolysis. Immunoblots of unlabeled phosphorylated reductase compared with gels of immunoprecipitated 32P-labeled reductase resolved a 52-56-kDa doublet which contained 32P solely in the upper band. These data suggest that a major phosphorylation site of HMG-CoA reductase lies within the 'linker' segment joining the membrane spanning and cytoplasmic domains of the native 97-kDa protein.

Original languageEnglish
Pages (from-to)4877-4887
Number of pages11
JournalJournal of Biological Chemistry
Volume264
Issue number9
StatePublished - 1989

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Phosphorylation
Liver
Rats
Oxidoreductases
Degradation
Enzymes
Coenzyme A
3-hydroxy-3-methylglutaryl-coenzyme A
Hepatocytes
Proteolysis
Mevalonic Acid
Calpain
Microsomes
Lysosomes
Immunoprecipitation
Joining
Peptide Hydrolases
Phosphotransferases

ASJC Scopus subject areas

  • Biochemistry

Cite this

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title = "Phosphorylation of native 97-kDa 3-hydroxy-3-methylglutaryl-coenzyme A reductase from rat liver. Impact on activity and degradation of the enzyme",
abstract = "Immunoprecipitation of native rat liver microsomal 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, phosphorylated by [γ-32P]ATP in the presence of reductase kinase, revealed a major 97-kDa 32P band which disappeared upon competition with pure unlabeled 53-kDa HMG-CoA reductase. A linear correlation between the expressed/total HMG-CoA reductase activity ratio (E/T) and the fraction of 32P released from the 97-kDa enzyme established the validity of the E/T ratio as an index of HMG-CoA reductase phosphorylation state in isolated microsomes. Incubation of rat hepatocytes with mevalonolactone resulted in a rapid increase in phosphorylation of microsomal reductase (decrease in E/T) followed by an enhanced rate of decay of total reductase activity which was proportional to the loss of 97-kDa enzyme mass determined by immunoblots. Inhibitors of lysosome function dampened both basal and mevalonate-induced reductase degradation in hepatocytes. In an in vitro system using the calcium-dependent protease calpain-2, up to 5-fold greater yields of soluble 52-56-kDa fragments of reductase (immunoblot and total activity) were obtained when the substrate 97-kDa reductase was phosphorylated before proteolysis. Immunoblots of unlabeled phosphorylated reductase compared with gels of immunoprecipitated 32P-labeled reductase resolved a 52-56-kDa doublet which contained 32P solely in the upper band. These data suggest that a major phosphorylation site of HMG-CoA reductase lies within the 'linker' segment joining the membrane spanning and cytoplasmic domains of the native 97-kDa protein.",
author = "Parker, {R. A.} and Steven Miller and Gibson, {D. M.}",
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T1 - Phosphorylation of native 97-kDa 3-hydroxy-3-methylglutaryl-coenzyme A reductase from rat liver. Impact on activity and degradation of the enzyme

AU - Parker, R. A.

AU - Miller, Steven

AU - Gibson, D. M.

PY - 1989

Y1 - 1989

N2 - Immunoprecipitation of native rat liver microsomal 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, phosphorylated by [γ-32P]ATP in the presence of reductase kinase, revealed a major 97-kDa 32P band which disappeared upon competition with pure unlabeled 53-kDa HMG-CoA reductase. A linear correlation between the expressed/total HMG-CoA reductase activity ratio (E/T) and the fraction of 32P released from the 97-kDa enzyme established the validity of the E/T ratio as an index of HMG-CoA reductase phosphorylation state in isolated microsomes. Incubation of rat hepatocytes with mevalonolactone resulted in a rapid increase in phosphorylation of microsomal reductase (decrease in E/T) followed by an enhanced rate of decay of total reductase activity which was proportional to the loss of 97-kDa enzyme mass determined by immunoblots. Inhibitors of lysosome function dampened both basal and mevalonate-induced reductase degradation in hepatocytes. In an in vitro system using the calcium-dependent protease calpain-2, up to 5-fold greater yields of soluble 52-56-kDa fragments of reductase (immunoblot and total activity) were obtained when the substrate 97-kDa reductase was phosphorylated before proteolysis. Immunoblots of unlabeled phosphorylated reductase compared with gels of immunoprecipitated 32P-labeled reductase resolved a 52-56-kDa doublet which contained 32P solely in the upper band. These data suggest that a major phosphorylation site of HMG-CoA reductase lies within the 'linker' segment joining the membrane spanning and cytoplasmic domains of the native 97-kDa protein.

AB - Immunoprecipitation of native rat liver microsomal 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, phosphorylated by [γ-32P]ATP in the presence of reductase kinase, revealed a major 97-kDa 32P band which disappeared upon competition with pure unlabeled 53-kDa HMG-CoA reductase. A linear correlation between the expressed/total HMG-CoA reductase activity ratio (E/T) and the fraction of 32P released from the 97-kDa enzyme established the validity of the E/T ratio as an index of HMG-CoA reductase phosphorylation state in isolated microsomes. Incubation of rat hepatocytes with mevalonolactone resulted in a rapid increase in phosphorylation of microsomal reductase (decrease in E/T) followed by an enhanced rate of decay of total reductase activity which was proportional to the loss of 97-kDa enzyme mass determined by immunoblots. Inhibitors of lysosome function dampened both basal and mevalonate-induced reductase degradation in hepatocytes. In an in vitro system using the calcium-dependent protease calpain-2, up to 5-fold greater yields of soluble 52-56-kDa fragments of reductase (immunoblot and total activity) were obtained when the substrate 97-kDa reductase was phosphorylated before proteolysis. Immunoblots of unlabeled phosphorylated reductase compared with gels of immunoprecipitated 32P-labeled reductase resolved a 52-56-kDa doublet which contained 32P solely in the upper band. These data suggest that a major phosphorylation site of HMG-CoA reductase lies within the 'linker' segment joining the membrane spanning and cytoplasmic domains of the native 97-kDa protein.

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