• YAKHAK HOEJI
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• v.37 no.2
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• pp.149-157
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• 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
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• v.35 no.4
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• pp.341-347
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• 1991

It was reported that protein carboxymethylation is involved in amylase secretion of parotid gland by isoproterenot. It was also suggested that a small part of the total cellular protein carboxymethylation is directly involved in pancreatic enzyme secretion. On the contrary, other authors reported that there is no relationship between protein carboxymethylation and secretion in pancreas and parotid gland. In recent study, it was proposed that a methyl acceptor protein plays a limited modulatory role in the coupling of cytosolic $Ca^{++}$ accumulation and exocytosis. In this study, the effects of cholinergic and adrenergic agents on the activities of protein methylase II in pancreatic tissues were examined to test the relationship between protein methylation and pancreatic secretion. The results are as follows. The activity of amylase was slightly increased at the concentration of $10^{-5}$ M of isoproterenol and norepinephrine. The activities of protein methylase I and II were decreased by isoproterenol and norepinephrine, but the activities of protein methylase III were hardly changed. The cholinergic stimulants acetylcholine and carbachol at a concentration of $10^{-5}$ M increased the activities of protein methylase I and decreased the activitiy of protein methylase III compared with control.

• YAKHAK HOEJI
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• v.35 no.6
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• pp.473-482
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• 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 $3.23{\times}10^{-5}M$.

• Korean Journal of Microbiology
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• v.24 no.2
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• pp.86-90
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• 1986

The DNA methylated by Hpa II methylase was not cleaved by Sma, I, Ava I and Nae I endonucleases. This experimental data could be interpreted as strong evidences that Sma I, Ava I and Nae I methylases which yet to be isolated would methylate on the inmost cytosine nucleotide within their hexameric recognition sequences. The facts that Sma I, Ava I and Nae I endonucleases can not cleave the DNA methylated by Hpa II methylase are the valuable informations for protecting DNAs upon cleavage reactions by Sma I, Ava I and NAe I endonucleases especially for cDNA insertion experiments into vector DNAs using Sma I, Ava I and Nae I oligonucleotide linkers. In the case of Xma I endonuclease, partially cleaved DNA fragments were observed although the reaction rate was greatly decreased. This result implies that the methylation site of Xma I methylase which yet to be isolated would not be the same as that of Hpa II methylase in Xma I sequence.

• BMB Reports
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• v.28 no.2
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• pp.149-154
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• 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 $1.38{\times}10^{-6}M$.

• BMB Reports
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• v.30 no.6
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• pp.410-414
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• 1997

We purified a protein carboxyl O-methyltransferase (protein methylase II) from porcine spleen to homogeneity. The molecular weight of the porcine spleen protein methylase II (ps-PM II) was estimated to be 27,500 daltons on SDS-PAGE. Amino acid sequence of N-terminal 28 residues for ps-PM II was identified. Amino-terminal three amino acid residues of ps-PM II were deleted when compared to those of other protein carboxyl methytransferase. S-Adenosyl-L-homocysteine competitively inhibits ps-PM II with a K, value of $1.63{\times}10^{-7}M$. Myelin basic protein exhibited the highest methyl-accepting capacity among the proteins tested.

• YAKHAK HOEJI
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• v.31 no.3
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• pp.173-181
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• 1987

Protein methylase I has been partially purified from hog pancreas with a 11% yeild. The final preparation is completely free of any other protein-specific methyltransferases and endogenous substrate proteins. The enzyme has an optimum pH of 7.2 and the approximate molecular weight is above 800 thousands dalton. The Km values for S-adenosyl-L-methionine and histone type II-A are 1.32$\times$10$^{-5}$M. The Ki value for S-adenosyl-L-homocysteine is 1.52$\times$10$^{-6}$M. The effect of enyzme concentration on the activity showed a slight sigmoidal curve suggesting the involvement of certain cofactors. Even though the purified enzyme showed two bands on polyacrylamide gel electrophoresis, the enzyme is highly specific for the arginine residues of protein and specifically, highly specific for histone, suggesting histonespecific protein methylase I.

• YAKHAK HOEJI
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• v.27 no.4
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• pp.295-301
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• 1983

Among the many protein modifications methylation is being investigated actively with regard to bacterial chemotaxis, gene regulation, muscle contraction, cytochrome c methylation, and the synthesis of the acyl transporter, carnitine. In this study the activities of protein methylase I, II, and III in pancreatic tissues of rat, mouse, and guinea pig were examined. Furthermore, the effect of cholinergic agents on the activity of protein methylases in pancreatic fragment of guinea pig was also examined in order to test the relationship between protein methylation and pancreatic secretion. The results are as follows. 1) The activities of protein methylases were generally high in pancreatic tissues of guinea pig and mouse but low in the tissue of rat. 2) The cholinergic stimulants, acetylcholine and carbachol at a concentration of $10^{-5}M$ decreased the activities of protein methylase I, II, and III compared with unstimulated control. 3) The inhibitory effect of the cholinergic stimulant on the activities of protein methylases was not blocked by atropine at a concentration of $10^{-5}M$.

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

• Archives of Pharmacal Research
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• v.22 no.3
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• pp.237-242
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• 1999

A proteinacious inhibitor with a molecular weight of 1,600 Da which inhibits S-adenosyl-L-methionine-dependent transmethylation reactions was purified from porcine liver to homogeneity by procedures including boiling, Sephadex G-25 column chromatography and repeated HPLC. Employing both Nuclear Magnetic Resonance (NMR) and Fast Atom Bombardment-Mass (FAB-Mass) spectroscopy, S-adenosylhomocysteine was conclusively identified as an integral part of the inhibitor. The purified S-adenosylhomocysteine was competitive with S-adenosyl-L-methionine with Ki value of $6.3{\times}10^{-6}$ M towards protein methylase II.