Identification of phosphorylation sites in glycine N-methyltransferase from rat liver.

Previous studies have shown that rat glycine N-methyltransferase (GNMT) is phosphorylated in vivo, and could be phosphorylated in vitro on serine residues with a significant increase of enzyme activity, but no phosphorylation sites were identified. In this work the identification of the specific phosphorylation sites of rat GNMT is reported. Three different preparations of rat GNMT were analyzed: (1) purified from liver by standard methods of protein purification, (2) prepared from isolated hepatocytes and from liver tissue by immunoprecipitation, and (3) recombinant protein expressed in Escherichia coli. We measured the molecular weights of protein isoforms using electrospray mass spectrometry and used liquid chromatography-tandem mass spectrometry (LC-MS/MS) of peptides resulting from tryptic and chymotryptic digests. We also performed chemical analysis of phosphoamino acids and protein sequencing. In all samples, the phosphorylated serine residues 71, 182, and 241 were found. In GNMT prepared from liver tissue and hepatocytes an S9 additional residue was found to be phosphorylated. In hepatocytes and in recombinant GNMT S139 was detected. Serine 9 was also identified as a target for cAMP-dependent protein kinase in vitro. The positions of these phosphorylated residues in the tertiary structure of GNMT indicate their possible effect on enzyme conformation and activity.

S-adenosylhomocysteine hydrolase deficiency in a human: a genetic disorder of methionine metabolism.

We report studies of a Croatian boy, a proven case of human S-adenosylhomocysteine (AdoHcy) hydrolase deficiency. Psychomotor development was slow until his fifth month; thereafter, virtually absent until treatment was started. He had marked hypotonia with elevated serum creatine kinase and transaminases, prolonged prothrombin time and low albumin. Electron microscopy of muscle showed numerous abnormal myelin figures; liver biopsy showed mild hepatitis with sparse rough endoplasmic reticulum. Brain MRI at 12.7 months revealed white matter atrophy and abnormally slow myelination. Hypermethioninemia was present in the initial metabolic study at age 8 months, and persisted (up to 784 microM) without tyrosine elevation. Plasma total homocysteine was very slightly elevated for an infant to 14.5-15.9 microM. In plasma, S-adenosylmethionine was 30-fold and AdoHcy 150-fold elevated. Activity of AdoHcy hydrolase was approximately equal to 3% of control in liver and was 5-10% of the control values in red blood cells and cultured fibroblasts. We found no evidence of a soluble inhibitor of the enzyme in extracts of the patient's cultured fibroblasts. Additional pretreatment abnormalities in plasma included low concentrations of phosphatidylcholine and choline, with elevations of guanidinoacetate, betaine, dimethylglycine, and cystathionine. Leukocyte DNA was hypermethylated. Gene analysis revealed two mutations in exon 4: a maternally derived stop codon, and a paternally derived missense mutation. We discuss reasons for biochemical abnormalities and pathophysiological aspects of AdoHcy hydrolase deficiency.