Dephosphorylation of L-pyruvate kinase during rat liver hepatocyte isolation.
In isolated rat liver cells, the inhibition of L-pyruvate kinase (L-PK) by a cyclic AMP-dependent phosphorylation mechanism is involved in the hormonal control of glycolysis and gluconeogenesis. The aim of this study was to ascertain whether or not the in vivo phosphorylation state of the enzyme was maintained during the liver perfusion used to prepare isolated liver cells. When the L-PK phosphorylation state was studied indirectly in liver extracts by kinetic measurement, it was found that, during the perfusion, the S0.5 of phosphoenol pyruvate (PEP) for L-PK was decreased in a time-dependent manner from 1 +/- 0.08 to 0.64 +/- 0.1 mM (P less than 0.01) and 0.58 +/- 0.06 mM in liver cells. This shift was prevented only by the addition of glucagon to the perfusion medium. The extent of phosphorylation of L-PK was also estimated by incubation of the liver extract with [gamma-32P]ATP, protein kinase, and cyclic AMP, and measurement of 32Pi incorporated in L-PK by specific immunoprecipitation. In liver extracts removed at the beginning of the perfusion, 0.4 mol Pi/mol L-PK was incorporated and there was no stimulation by cyclic AMP. In contrast, in the liver extracts removed after 30 min of perfusion, cyclic AMP stimulated 32P incorporation two to threefold, and 1.6 mol Pi/mol L-PK was incorporated. These data suggest that L-PK was activated by a dephosphorylation mechanism during rat liver perfusion. This phenomenon could be involved in the classical inactivation of gluconeogenesis observed in the perfused rat liver model.
Riou
J P
JP
Audigier
C
C
Laville
M
M
Beylot
M
M
Pigeon
P
P
Mornex
R
R
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002469:Cell Separation / Q000379:methods*; D011232:Chemical Precipitation; D000242:Cyclic AMP / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005934:Glucagon / Q000494:pharmacology; D007120:Immunochemistry; D007700:Kinetics; D008099:Liver / Q000166:cytology / Q000201:enzymology*; D008297:Male; D010477:Perfusion; D010728:Phosphoenolpyruvate / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D011770:Pyruvate Kinase / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains
D010728:Phosphoenolpyruvate; D005934:Glucagon; D000242:Cyclic AMP; D011494:Protein Kinases; D011770:Pyruvate Kinase; D002118:Calcium