Transport of rat liver glycine N-methyltransferase into rat liver nuclei.
Rat liver cytosolic glycine N-methyltransferase (GNMT) catalyzes the S-adenosylmethionine-dependent methylation of glycine to sarcosine. It is comprised of four identical 292-amino acid residue subunits. Recently, evidence has been provided to show that GNMT is identical to the cytosolic receptor for benzo[a]pyrene, which induces cytochrome P450 1A1 gene expression. In the present study we show that chemical modification of purified rat liver GNMT with fluorescein isothiocyanate (FITC) resulted in dissociation of the tetrameric enzyme and was accompanied by loss of enzyme activity. Amino acid sequence analysis of the FITC-labeled peptides obtained by hydrolysis of the modified protein with Staphylococcus aureus V8 protease revealed that lysines 45, 89, 92, 96, 122, and 147 were modified. Lys-122 and Lys-147 were derivatized in tetrameric, dimeric, and monomeric forms of the enzyme. Lysines 45, 89, 92, and 96 were derivatized only in monomeric GNMT, suggesting that modification of these residues resulted in GNMT dissociation. The modified monomeric GNMT was quickly transported into isolated rat liver nuclei. This transport was specific for the GNMT monomer, since neither tetramer nor dimer was able to enter the nuclei. Bovine carbonic anhydrase, similar in size to the GNMT monomer, was labeled with FITC to a similar extent but was not transported into the nuclei. Disruption of the nuclei containing fluorescein-labeled GNMT and subsequent extraction of the nuclear lysate with both high and low salt buffers recovered FITC-GNMT only in the chromatin pellet. Our study supports the suggestion of an additional function for GNMT, probably connected with regulation of cytochrome P450 1A1 gene expression.
Krupenko
N I
NI
Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, USA.
Wagner
C
C
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D002256:Carbonic Anhydrases / Q000378:metabolism; D002417:Cattle; D002467:Cell Nucleus / Q000201:enzymology*; D002850:Chromatography, Gel; D003198:Computer Simulation; D050938:Glycine N-Methyltransferase; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008239:Lysine; D046911:Macromolecular Substances; D008297:Male; D008780:Methyltransferases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008961:Models, Structural; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010449:Peptide Mapping; D011487:Protein Conformation; D051381:Rats; D017207:Rats, Sprague-Dawley
D000906:Antibodies; D046911:Macromolecular Substances; D010446:Peptide Fragments; D008780:Methyltransferases; D050938:Glycine N-Methyltransferase; C494512:Gnmt protein, rat; D002256:Carbonic Anhydrases; D008239:Lysine