• YAKHAK HOEJI
• /
• v.37 no.2
• /
• pp.149-157
• /
• 1993

Protein methylase inhibitor which is a modulator of biological methylation has been purified and characterized from porcine liver soluble fraction by cell fractionation, Sephadex G25 chromatography, reverse phase HPLC, size exclusion HPLC. The results are summarized as follows. 1) The purified inhibitor shows apparent homogeneity, as judged by HPLC. 2) A molecular weight of the purified inhibitor which is composed of 18 amino acid residues is about 1,400 daltons. 3) A single absorption peak of ultraviolet spectrum was observed at 260nm. 4) The inhibitor was not inactivated by heating at $100^{\circ}C$ until 60min. and its activity was not influenced by treatment with digestive enzymes, such as trypsin, pepsin, pronase, chymotrypin, lysozyme, DNase, and RNase. 5) The purified inhibitor inhibited protein rnethylase I, II, III and phospholipid methyltransferase activities. 6) The purified inhibitor inhibited noncompetitively protein methylase II from porcine liver, spleen, and testis. 7) The $K_{i}$ values for protein methylase II from porcine liver, spleen, and testis were 300nM, 250nM, 297nM, respectively.

• YAKHAK HOEJI
• /
• v.35 no.6
• /
• pp.473-482
• /
• 1991

Protein methylase II (S-adenosyl-L-methionine:protein carboxyl-O-methyltransferase; EC 2.1.1.24., PM II) was purified from chicken pancreas by subcellular fractionation, DEAE-cellulose chromatography, QAE-Sephadex A-50 chromatography, Sephadex G-75 chromatography, and Sephadex G-75 rechromatography. The purified PM II gave a single band upon polyarcrylamide gel electrophoresis both in the presence of SDS and in Tris glycine buffer without SDS. The pI value of purified PM II was identified as 5.7 on isoelectric focusing gel. Properties and activities of PM II were studied and the following results were obtained. 1) PM II from chicken pancreas was purified approximately 221-fold with a yield of 1.3%. 2) The purified PM II appear constituted of a single polypeptide chain of a molecular weight 46,800 daltons. 3) Hemoglobin exhibited the highest of methyl-accepting activity among the substrates tested. 4) The purified PM II has a $K_m$ of $4.67{\times}10^{-6}M$ and a $V_{max}$ of 37.5 pmoles of $methyl-^{14}C/min./mg$ enzyme for $SAM^{-14}CH_3$ as methyl donor in the presence of histone type II-As. 5) It is found that S-adenosyl-L-homocysteine is a competitive inhibitor for PM II with $K_i$ value of $3.23{\times}10^{-5}M$.

• Journal of Life Science
• /
• v.8 no.1
• /
• pp.109-117
• /
• 1998

The synthesis and methylation in vivo of myleline basic protein(MBP) during the mouse brain devlopment was found to be the highest in youngest brain and declined progressively in mature brains. The relative rate of protein synthesis and methylation was a maximal ration in the youngest brain, This high ratio was wdll correlated with the higher protein methylase I (PM I) activity in younger brains. The jimpy mouse is the most severely affected dysmyelinating mutant and is characterized by failure to incorporate MBP into myelin. sheath. The MBP-specific PM I activity in 15-, 18-, and 21-days old hemizygous jimpy mice(jp/y)brains decreased by 20, 50 and 75%, respectively. Myelin fraction with different degrees of compaction were isolated from bovine brain, the most compact myelin fraction exhibited higher methylaccepting activity than the less compact dense fractions.

• BMB Reports
• /
• v.28 no.2
• /
• pp.149-154
• /
• 1995

Protein methylase II (S-adenosyl-L-methionine : protein O-methyl-transferase, EC 2.1.1.24; PM II) was purified approximately 1250-fold from porcine testis by fractional precipitation and DEAE-cellulose chromatography, followed by gel filtration on a Sephadex G-75 column and HPLC on a Protein Pak 125 column. The molecular weight of the enzyme was estimated to be 33,000 daltons by SDS-PAGE, which agreed with the value determined by gel filtration. Isoelectric focusing of purified PM II showed a single protein species with an isoelectric point of 6.2. The optimum pH for the reaction was 6.0. The $K_m$ value of the enzyme was $1{\times}10^{-5}M$ with a $V_{max}$ value of 769 pmol/min/mg of enzyme. S-adenosyl-L-homocysteine is a competitive inhibitor of PM II with a $K_i$ value of $1.38{\times}10^{-6}M$.

• Biomolecules & Therapeutics
• /
• v.2 no.1
• /
• pp.47-53
• /
• 1994

The methylation response as well as the level of methyl donor substance, 5-adenosyl-L-methionine (SAM) has been suggested to be related to hepatotoxicity including hepatocarcinogenesis. But direct correlation between protein methylation and hepatotoxicity has not been established to the present. To observe relationship between protein methylation and short-term hepatotoxicity induced by chemical substances, the activities of protein methylase I and II (PM I, PM II) were examined in cytosolic fraction of SD rat treated orally with acetaminophen(AA), $\alpha$-naphtyl-isothiocyanate (ANIT) and tetracycline (TC) that was known to produce necrosis, cholestasis and steatosis respectively. To evaluate the degree of hepatotoxicity induced by each chemicals, we observed the serum levels of indicative parameters and histopathological alteration. In AA treated group, the activities of PM I were increased at 6, 12 hours after administration, prior to the appearance of the hepatotoxicity by clinical parameters. It was suggested that the levels of PM I were related with the initial stage of hepatotoxic mechanism induced by AA. In ANIT treated group, though most of clinical parameters were significantly increased at 24, 48 hours after administration, the activity of PM I was not changed, indicating that ANIT induced hepatotoxicity was not coupled to protein methylation.