J. Biol. Chem., Vol. 263, Issue 23, 11211-11216, 08, 1988
Complete purification and immunochemical analysis of S- adenosylmethionine synthetase from bovine brain
K Mitsui, H Teraoka and K Tsukada
Department of Pathological Biochemistry, Tokyo Medical and Dental University, Japan.
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.
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