S-adenosyl-L-methionine:L-methionine S-methyltransferase from germinating barley. Purification and localization.
S-Adenosyl-L-methionine:L-methionine S-methyltransferase (MMT) catalyzes the synthesis of S-methyl-L-methionine (SMM) from L-methionine and S-adenosyl-L-methionine. SMM content increases during barley (Hordeum vulgare L.) germination. Elucidating the role of this compound is important from both a fundamental and a technological standpoint, because SMM is the precursor of dimethylsulfide, a biogenic source of atmospheric S and an undesired component in beer. We present a simple purification scheme for the MMT from barley consisting of 10% to 25% polyethylene glycol fractionation, anion-exchange chromatography on diethylaminoethyl-Sepharose, and affinity chromatography on adenosine-agarose. A final activity yield of 23% and a 2765-fold purification factor were obtained. After digestion of the protein with protease, the amino acid sequence of a major peptide was determined and used to produce a synthetic peptide. A polyclonal antibody was raised against this synthetic peptide conjugated to activated keyhole limpet hemocyanin. The antibody recognized the 115-kD denatured MMT protein and native MMT. During barley germination, both the specific activity and the amount of MMT protein increased. MMT-specific activity was found to be higher in the root and shoot than in the endosperm. MMT could be localized by an immunohistochemical approach in the shoot, scutellum, and aleurone cells but not in the root or endosperm (including aleurone).
Pimenta
M J
MJ
Plant Hormone Function, Frontier Research Program, Saitama, 351-0198, Japan. pimenta@postman.riken.go.jp
Kaneta
T
T
Larondelle
Y
Y
Dohmae
N
N
Kamiya
Y
Y
D000595:Amino Acid Sequence; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D018525:Germination; D001467:Hordeum / Q000201:enzymology* / Q000254:growth & development; D007150:Immunohistochemistry; D008780:Methyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data
D008780:Methyltransferases; C093871:methionine S-methyltransferase