Phosphorylation of beta-glucuronidases from human normal liver and hepatoma by cAMP-dependent protein kinase.
beta-Glucuronidases purified from human hepatoma and from normal liver could serve as a substrate for a cAMP-dependent protein kinase. The rate of phosphorylation reaction of the hepatoma beta-glucuronidase was rapid, whereas that of the normal liver beta-glucuronidase was slow and much lower. Stoichiometry of phosphorylation was 4.3 and 0.46 mol of phosphate/mol of the beta-glucuronidase from the hepatoma and normal liver, respectively. Tryptic peptide mapping of 32P-labeled beta-glucuronidase from hepatoma identified two distinct phosphopeptides (X and Y). The peptide from hepatoma hydrolase was phosphorylated predominantly at the X, while the peptide Y was the major phosphopeptide in the hydrolase of normal liver. Analysis of phosphoamino acids revealed two sites, phosphoserine and phosphothreonine. beta-Glucuronidase from hepatoma consisted of a major subunit with molecular mass of 64,000 (64 kDa) and a minor subunit with 76 kDa, whereas the hydrolase from normal liver had almost exclusively 64 kDa subunit. 32P-labeled beta-glucuronidase indicated that the 64 kDa subunit was phosphorylated both in hepatoma and normal liver beta-glucuronidases.
Ono
M
M
Biochemistry Laboratory, Hokkaido University School of Medicine, Sapporo, Japan.
Taniguchi
N
N
Makita
A
A
Fujita
M
M
Sekiya
C
C
Namiki
M
M
D000596:Amino Acids / Q000032:analysis; D006528:Carcinoma, Hepatocellular / Q000201:enzymology*; D000242:Cyclic AMP / Q000494:pharmacology*; D005966:Glucuronidase / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D005779:Immunodiffusion; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008113:Liver Neoplasms / Q000201:enzymology*; D008970:Molecular Weight; D010446:Peptide Fragments / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D010769:Phosphothreonine / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism*; D014357:Trypsin
D000596:Amino Acids; D010446:Peptide Fragments; D010710:Phosphates; D010769:Phosphothreonine; D010768:Phosphoserine; D000242:Cyclic AMP; D011494:Protein Kinases; D005966:Glucuronidase; D014357:Trypsin