Molecular cloning, sequence analysis, and expression in Escherichia coli of the cDNA for guanidinoacetate methyltransferase from rat liver.
Five cDNA clones encoding rat liver guanidinoacetate methyltransferase (S-adenosyl-L-methionine: guanidinoacetate N-methyltransferase, EC 2.1.1.2) were isolated from a lambda gt11 cDNA library by use of a polyclonal antibody to the purified enzyme. Sequence analysis of the longest cDNA indicated that it consisted of 711 base pairs (bp) of coding region, 51 bp of 5' noncoding region, and 162 bp of 3' noncoding region excluding the poly(A) tail. The amino acid sequence deduced from the cDNA contained the sequences of NH2-terminal and three tryptic peptides. The predicted amino acid composition and molecular weight were in excellent agreement with those obtained with the purified enzyme. Introduction of the cDNA into plasmid pUC118 having the lac promoter resulted in a production in Escherichia coli of a Mr 26,000 polypeptide in the presence of isopropyl beta-D-thiogalactopyranoside. This protein represented as much as 5% of the bacterial soluble protein and showed the guanidinoacetate methyltransferase activity. Sequence analysis and tryptic peptide mapping indicated that the enzyme obtained by the recombinant DNA procedures was structurally identical to the liver enzyme, except for the absence of the NH2-terminal blocking group. Also, the enzyme showed kinetic properties indistinguishable from those of the liver enzyme.
Ogawa
H
H
Department of Biochemistry, Toyama Medical and Pharmaceutical University, Faculty of Medicine, Japan.
Date
T
T
Gomi
T
T
Konishi
K
K
Pitot
H C
HC
Cantoni
G L
GL
Fujioka
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D050937:Guanidinoacetate N-Methyltransferase; D008099:Liver / Q000201:enzymology; D008780:Methyltransferases / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
D011994:Recombinant Proteins; D004247:DNA; D008780:Methyltransferases; C494508:Gamt protein, rat; D050937:Guanidinoacetate N-Methyltransferase