Complete purification and immunochemical analysis of S-adenosylmethionine synthetase from bovine brain.
We have purified S-adenosylmethionine (AdoMet) synthetase about 3000-fold from bovine brain extract. The Km values of the enzyme for L-methionine and ATP were 10 and 50 microM, respectively. An apparent molecular mass of the enzyme was estimated to be 160 kDa by gel filtration on a Sephacryl S-200 column. Sucrose density gradient centrifugation gave a sedimentation coefficient of 8 S. Polyacrylamide gel electrophoresis of the purified enzyme in native system revealed a single protein band, whereas two polypeptide bands with molecular masses of 48 kDa (p48) and 38 kDa (p38) were observed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme. Antibody against bovine brain AdoMet synthetase was prepared by injecting the purified enzyme into a rabbit. Immunoblot analysis revealed that the antibody recognized both p48 and p38 in the impure enzyme preparations from bovine brain as well as in the purified enzyme. Specific antibodies against p48 and p38 were separated from the immunoglobulin fraction by an affinity purification, both of which inhibited the enzyme activity. These results indicate that AdoMet synthetase from bovine brain consists of two different polypeptides, p48 and p38.
Mitsui
K
K
Department of Pathological Biochemistry, Tokyo Medical and Dental University, Japan.
Teraoka
H
H
Tsukada
K
K
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D002499:Centrifugation, Density Gradient; D004591:Electrophoresis, Polyacrylamide Gel; D007150:Immunohistochemistry; D007700:Kinetics; D008715:Methionine / Q000378:metabolism; D008716:Methionine Adenosyltransferase / Q000302:isolation & purification*; D008970:Molecular Weight; D014166:Transferases / Q000302:isolation & purification*
D000255:Adenosine Triphosphate; D008715:Methionine; D014166:Transferases; D008716:Methionine Adenosyltransferase