骨格筋 S-Adenosyl-L-Methionine : Protein-Carboxyl O-Methyltransferase의 精製 및 特性 Protein-Carboxyl O-Methyltransferase from Bovine Skeletal Muscle = Purification and Characterization of S-Adenosyl-L-Methionine

다국어 초록 (Multilingual Abstract)

S-Adenosyl-L-methionine : protein-carboxyl 0-methyltransferase(prorein methylase II) has been purified from bovine skeletal muscle approximately 6,600-fold with a 7% yield by combination of ammonium sulfate precipitate, Sephadex G-75 chromatography, S-adenosyl-L-homocysteine-Sepharose 4B chromatography and hydroxyapatite chromatography, and then its characterizations and possible roles were investigated.
The enzyme showed a optimum pH around 6.0. It was heat labile, inactivated by heat-treatment at 60℃ for 6 minutes, and when stored at -20℃ in the presence 10% glycerol, its activity has almost stabilized in 6 months.
Copper ion(Cu^2+)and zinc ion(Zn^2+)were a potent inhibitors, the activity being completely inhibited at 0.25 mM and 1 mM respectively, The inhibition of copper and zinc ions were recovered 94%, 75% of its activity by adding 4 mM of EDTA, respectively.
The apparent Km value for S-adenosyl--L-methionine was 2.86 x 10^-6 M. Kinetic analysis of enzyme in the presence of 30 uM copper ion showed that the nature of the inhibition to the enzyme was noncompetitive considering that Km was constant and Vmax was decreased.
Histone IIA, immunoglobulin A and trypsin inhibitor were relatively good substrates for the enzyme.
The enzyme activity was completely inhibited by 0.5 mM p-hydroxymercuribenzoic acid, but all of the activity was recovered in adding 10 mM mercaptoethanol, and the molecular weight of the enzyme was 24,000.
Sarcolemma and sarcoplasmic reticulum of the skeletal muscle were good substrates for this enzyme and ^3H-CH_3 group was incorporated to 0.63 and 0.96 pmoles/mg protein/min, respectively.
The carboxylmethylation of the sarcolemma of the skeletal muscle occurred in the 5 protein fractions(M. W. : 45,000, 22,000, below 14,400etc.) and that of sarcoplasmic reticulum occurred in the 7 protein fractions (M. W. : 100,000, 45,000, 22,000, below 14, 400 etc) by SDS/polyacrylamide gel electrophoresis. Among these molecular weight 22,000 was strongly methylated.
These results discuss that protein methylase Il may play some roles in regulating physiological function of sarcolemma and sarcoplasmic reticulum of skeletal muscle.