Guanidoacetate methyltransferase from rat liver: purification, properties, and evidence for the involvement of sulfhydryl groups for activity.
Guanidoacetate methyltransferase (EC 2.1.1.2) has been purified about 800-fold from rat liver. The purified preparation shows a single protein band on polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. The molecular weight of the enzyme is estimated to be 25,000 and 26,000 by Sephadex gel molecular-exclusion chromatography and by electrophoresis in polyacrylamide gradient gel, respectively. The sodium dodecyl sulfate-denatured enzyme also has a molecular weight of 26,000; thus, the enzyme is a monomeric protein. Guanidoacetate methyltransferase as isolated is catalytically inactive, but is readily reactivated by incubation with a thiol. The reactivated enzyme, which contains 3 mol of sulfhydryl groups/mol of enzyme, is again inactivated by oxidized glutathione. This inactivation is accompanied by the disappearance of two sulfhydryl residues. The relationship between the loss of enzyme activity and the number of residues disappeared indicates that the integrity of these sulfhydryl residues is critical for activity. The oxidized enzyme fails to bind the substrate S-adenosylmethionine as evidenced by the equilibrium dialysis study. Alkylation of the nonoxidizable sulfhydryl by N-ethylmaleimide shows that this residue is also essential for activity. UV absorption, fluorescence, and CD spectra show no difference between the reduced and oxidized enzymes, but the former is more susceptible to proteolytic attack by trypsin. The enzyme has an isoelectric pH of 5.3, and is most active at pH 9.0. From the CD spectrum, an alpha helix content of 15% is calculated. The Km values for guanidoacetate and S-adenosylmethionine are 97.5 and 6.73 microM, respectively, at pH 8.0 and 37 degrees C.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D005978:Glutathione / Q000494:pharmacology; D005998:Glycine / Q000031:analogs & derivatives / Q000494:pharmacology; D050937:Guanidinoacetate N-Methyltransferase; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008780:Methyltransferases / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D051381:Rats; D011919:Rats, Inbred Strains; D012436:S-Adenosylmethionine / Q000494:pharmacology
D000596:Amino Acids; D012436:S-Adenosylmethionine; D008780:Methyltransferases; D050937:Guanidinoacetate N-Methyltransferase; D005978:Glutathione; C004946:glycocyamine; D005998:Glycine