Document Detail

Okadaic acid-induced, naringin-sensitive phosphorylation of glycine N-methyltransferase in isolated rat hepatocytes.
MedLine Citation:
PMID:  12697024     Owner:  NLM     Status:  MEDLINE    
Glycine N-methyltransferase (GNMT) is an abundant cytosolic enzyme that catalyses the methylation of glycine into sarcosine, coupled with conversion of the methyl donor, S -adenosylmethionine (AdoMet), into S -adenosylhomocysteine (AdoHcy). GNMT is believed to play a role in monitoring the AdoMet/AdoHcy ratio, and hence the cellular methylation capacity, but regulation of the enzyme itself is not well understood. In the present study, treatment of isolated rat hepatocytes with the protein phosphatase inhibitor okadaic acid, was found to induce an overphosphorylation of GNMT, as shown by proteomic analysis. The analysis comprised two-dimensional gel electrophoretic separation of (32)P-labelled phosphoproteins and identification of individual protein spots by matrix-assisted laser-desorption ionization-time-of-flight mass spectrometry. The identity of GNMT was verified by N-terminal Edman sequencing of tryptic peptides. Chromatographic separation of proteolytic peptides and (32)P-labelled amino acids suggested that GNMT was phosphorylated within a limited region, and only at serine residues. GNMT phosphorylation could be suppressed by naringin, an okadaic acid-antagonistic flavonoid. To assess the possible functional role of GNMT phosphorylation, the effect of okadaic acid on hepatocytic AdoMet and AdoHcy levels was examined, using HPLC separation for metabolite analysis. Surprisingly, okadaic acid was found to have no effect on the basal levels of AdoMet or AdoHcy. An accelerated AdoMet-AdoHcy flux, induced by the addition of methionine (1 mM), was likewise unaffected by okadaic acid. 5-Aminoimidazole-4-carboxamide riboside, an activator of the hepatocytic AMP-activated protein kinase, similarly induced GNMT phosphorylation without affecting AdoMet and AdoHcy levels. Activation of cAMP-dependent protein kinase by dibutyryl-cAMP, reported to cause GNMT phosphorylation under cell-free conditions, also had little effect on hepatocytic AdoMet and AdoHcy levels. Phosphorylation of GNMT would thus seem to play no role in regulation of the intracellular AdoMet/AdoHcy ratio, but could be involved in other GNMT functions, such as the binding of folates or aromatic hydrocarbons.
Michael T N Møller; Hamid R Samari; Monica Fengsrud; Per E Strømhaug; Anne C øStvold; Per O Seglen
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  The Biochemical journal     Volume:  373     ISSN:  0264-6021     ISO Abbreviation:  Biochem. J.     Publication Date:  2003 Jul 
Date Detail:
Created Date:  2003-07-04     Completed Date:  2003-08-15     Revised Date:  2009-11-18    
Medline Journal Info:
Nlm Unique ID:  2984726R     Medline TA:  Biochem J     Country:  England    
Other Details:
Languages:  eng     Pagination:  505-13     Citation Subset:  IM    
Department of Cell Biology, Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway.
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MeSH Terms
Aminoimidazole Carboxamide / analogs & derivatives*,  pharmacology
Bucladesine / pharmacology
Cell-Free System
Enzyme Inhibitors / pharmacology*
Flavonoids / pharmacology*
Glycine / metabolism*
Glycine N-Methyltransferase
Hepatocytes / drug effects*,  metabolism
Methyltransferases / metabolism*
Okadaic Acid / pharmacology*
Peptide Fragments / chemistry
Ribonucleosides / pharmacology
S-Adenosylhomocysteine / metabolism*
S-Adenosylmethionine / metabolism*
Reg. No./Substance:
0/Enzyme Inhibitors; 0/Flavanones; 0/Flavonoids; 0/Peptide Fragments; 0/Ribonucleosides; 10236-47-2/naringin; 2627-69-2/acadesine; 29908-03-0/S-Adenosylmethionine; 360-97-4/Aminoimidazole Carboxamide; 362-74-3/Bucladesine; 56-40-6/Glycine; 78111-17-8/Okadaic Acid; 979-92-0/S-Adenosylhomocysteine; EC 2.1.1.-/Methyltransferases; EC protein, human; EC N-Methyltransferase; EC protein, rat

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