Phosphorylation of hepatic phenobarbital-inducible cytochrome P-450.
The major phenobarbital-inducible cytochrome P-450 purified from rat liver, a member of family II of the cytochrome P-450 gene superfamily, is rapidly phosphorylated by cAMP-dependent protein kinase. The phosphorylation reaches greater than 0.5 mol phosphate/mol P-450 after 5 min and is accompanied by a decrease in enzyme activity. The serine residue in position 128 was shown to be the sole phosphorylation site and a conformational change of the protein was indicated by a shift of the carbon monoxide difference spectrum of the reduced cytochrome from 450 to 420 nm. Comparison of amino acid sequences of various cytochrome P-450 families revealed a highly conserved arginine residue in the immediate vicinity of the phosphorylated serine residue which constitutes the kinase recognition sequence. It also revealed that only the members of the cytochrome P-450 family II carry this kinase recognition sequence. To find out whether this phosphorylation also occurs in vivo, the exchangeable phosphate pool of intact hepatocytes derived from phenobarbital-pretreated rats was labeled with 32Pi followed by an incubation of the cells with the membrane-permeating dibutyryl-cAMP or with the adenylate cyclase stimulator glucagon to activate endogenous kinase. As a result, a microsomal polypeptide with the same electrophoretic mobility as cytochrome P-450 became strongly labeled. Peptide mapping and immunoprecipitation with monospecific antibodies identified this protein as the major phenobarbital-inducible cytochrome P-450. It becomes phosphorylated at the same serine residues as in the cell-free phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)
Pyerin
W
W
German Cancer Research Center, Institute of Experimental Pathology, Heidelberg, FRG.
Taniguchi
H
H
D000818:Animals; D002478:Cells, Cultured; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004790:Enzyme Induction; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008297:Male; D010449:Peptide Mapping; D010634:Phenobarbital / Q000494:pharmacology*; D010766:Phosphorylation; D014176:Protein Biosynthesis; D011487:Protein Conformation; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains
D007527:Isoenzymes; D003577:Cytochrome P-450 Enzyme System; D011494:Protein Kinases; D010634:Phenobarbital