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1 J. Exp. Biomed. Sci. 11 (2005) Catechol-O-Methyltransferase Activity from Regenerating Liver after Partial Hepatectomy in Rats You-Hee Kim, Hye-Jung Choi and Chun-Sik Kwak Department of Biochemistry, Keimyung University, School of Medicine, Daegu , Korea The change of catechol-o-methyltransferase (COMT) activity during regeneration of rat liver was studied. Cytosolic, mitochondrial and microsomal COMTs activities were estimated in regenerating rat livers over a period of ten s after 70% (median and left lateral lobes) partial hepatectomy. The values of Km and Vmax in the hepatic enzymes were also measured. The activities of cytosolic and microsomal COMTs in regenerating rat liver after partial hepatectomy were found to be significantly increased between the second and the third. Whereas the mitochondrial COMT activity did not change. The Vmax values of the cytosolic and microsomal COMTs in the regenerating rat liver were significantly increased at the second after partial hepatectomy, however, the Km values of the above hepatic enzymes did not vary in all the experimental groups. Therefore, the results suggest that the biosynthesis of COMT was increased during the regeneration of rat liver. Key Words: Catechol-O-methyltransferase, Partial hepatectomy, Regenerating liver 서 Catechol-O-methyltransferase (S-adenosyl-L-methionine: catechol-o-methyltransferase, EC , COMT) 는 catechol 및 catecholamine에 S-adenosyl-L-methionine으로부터 methyl 기를이전받아 catechol 또는 catecholamine을메틸화시키는반응을촉매하는제 2상생체이물생체변환 (phase 2 xenobiotic biotransformation) 효소로서 (Kim, 1979; Borchardt, 1980) 주로간세포의세포질, 미토콘드리아및내형질세망에국재되어있다 (Borchardt, 1980; Raxworthy et al., 1982; Mun, 1996). 간이손상을받으면간은손상을수복하기위하여재생이활발해진다 (Matsumoto et al., 1991). 그러나간의재생에대한생화학적지견은충분치않다. 재생간의생화학적연구를 위해서는쥐의간엽을부분절제하여형성된재생간을실험적모델로이용하고있다. 쥐의간엽들중일부간엽을절제하면남은간엽은급격히재생되어비대해지며 (Lieberman et al., 1965), 재생이왕성한시기의재생간에서는대사의속도를조절하기위하여여러효소들의활성도가변동된다. 특히재생간에서활성도가변동되는효소들중에서는생체이물 * 논문접수 : 2004년 12월 27일수정재접수 : 2005년 2월 25일 교신저자 : 곽춘식, ( 우 ) 대구광역시중구동산동 194번지, 계명대학교의과대학생화학교실 Tel: , Fax: kwak@dsmc.or.kr 론 생체변환효소들의활성도가변동이심하다 (Mun et al., 1988; Kwak et al., 1989; Joo et al., 2002) 고한다. COMT도생체이물생체변환효소로서간에주로존재하는효소인만큼재생간에서는이효소의활성도가변동될것으로생각된다. 그러나이에대한보고는찾아볼수없었다. 따라서재생간에서이효소의활성도변동과그기전을알아냄으로써간재생시간의생체이물생체변환기능의일단을파악할수있을것으로생각된다. 이연구는재생간에서 COMT의활성도변동을알아보기위하여시행한연구로써쥐간의중엽과좌측외엽을절제한후 12시간부터 10일까지간의세포질, 미토콘드리아및마이크로솜에서이효소의활성도를측정하였으며아울러쥐의간엽을절제한후 2일경과시킨쥐간에서이효소의 Km값및 Vmax값도함께측정하여이들성적을보고하고자한다. 재료및방법 1. 시약 S-(5'-Adenosyl)-L-methionine iodide, 3, 4-dihydroxybenzoic acid, DL-dithiothreitol, Triton X-100, catechol-o-methyltransferase (from porcine liver, C 1897) 및단백질표준액 (10 g/100 ml bovine serum albumin) 등은 Sigma사 ( 미국 ) 제품을사용하였으며 [methyl- 3 H] S-adenosyl-L-methionine은 New England Nuclear사 ( 미국 ) 의제품을그리고 PPO (2,5-diphenyloxazole),

2 bis-msb {ρ-bis-(o-methylstyryl benzene)}, toluene (scintillation grade) 등은 Packard사 ( 미국 ) 의제품을사용하였다. 그외일반시약은시판되는특급또는일급품을사용하였다. 2. 동물및처치동물은 4주이상같은조건으로사육한체중 320~350 g이되는 Sprague-Dawley종의숫쥐를사용하였으며한군을 5마리로하여다음과같이 12개군으로나누었다. 1) 가수술군가수술후 12시간, 1일, 2일, 3일, 6일및 10일에희생시킨군 ( 총 6군 ). 2) 간엽절제수술군간엽절제수술후 12시간, 1일, 2일, 3일, 6일및 10일에희생시킨군 ( 총 6군 ), 각실험군은개별분리수용하였으며실험전후에일정한조건으로사육하였다. 사료는시판되는삼양유지사료주식회사제품인실험동물사료를먹도록하였다. 간엽절제수술및가수술은효소활성의일중변동을고려하여쥐를오후 2시에서 5시사이에희생시킬수있도록수술시간을조절하였으며 12시간금식시킨후 ether 마취하에서실시하였다. 쥐간엽절제수술은복부정중선을따라상복부를약 2 cm 절개하여간의중엽과좌측외엽을복강밖으로압출하고인접조직사이의인대를절단한후간엽의기저부위를결찰한뒤간엽을절제하였다. 절제한간엽은전체간의약 70% 가되며이것을원래간 (original liver) 이라부르기로하였다. 가수술은단순개복술만시행하였다. 3. 간적출및세포분획모든실험군에서간의적출은 12시간금식시킨후 ether 마취하에서시행하였으며복부대동맥으로부터채혈하여쥐를실혈사시켰다. 그리고는간문맥에삽관한후 4 의 0.25 M sucrose액으로관류하여간에남아있던혈액을제거한다음간을적출하였다. 또한절제한원래간은 4 의 0.25 M sucrose액으로잘씻어서간에남아있던혈액을가능한한모두제거하였다. 그리고적출한간은면포로균등히압박하여간에남아있던 sucrose액을가능한한모두제거하였다. 간의세포분획은절제한간엽과적출한간들을즉시 2~ 4 로냉각한후잘게썰어서절편으로만들고혼합하여그중약 5 g을취하여 9배량의 0.25 M sucrose액을넣은다음 Teflon pestle glass homogenizer (chamber clearance 0.005~0.007 inches, Thomas사, 미국 ) 로 2~4 를유지하면서 400 rpm의속도로조심스럽게 5회왕복마쇄하여 10% (w/v) 의간조직균질액을만들었다. 이간균질액모두를취하여 sucrose density gradient 원심분리법 (Kwak et al., 1986) 으로세포질, 미토콘드리아및마이크로솜분획을분리하였다. 위의세포분획 법에서모든조작은 2~4 에서시행하였으며이때사용한원심분리기는 Du Pont Sorvall사 ( 미국 ) 의 RC-5B refrigerated superspeed centrifuge와 OTD-65B ultracentrifuge였다. 이때사용한 rotor는 Du Pont Sorvall사의 SS-34 및 T865 rotor였고 sucrose linear density gradi-ent용액의제조는 gradient former (model 570, ISCO, 미국 ) 를사용하였다 4. 효소시료조제 COMT 활성도측정용효소시료의조제는분리한마이크로솜분획및미토콘드리아분획 4.5 ml에대하여 0.5 ml의 sucrose-triton X-100 (Triton X ml와 sucrose 8.56 g을증류수에녹여 100 ml 만든다 ) 액을넣어 4 에서 30분간방치한후이액을이효소활성도측정용시료로사용하였으며세포질분획은아무런처치없이원액그대로사용하였다. 5. 효소활성도측정간세포질, 미토콘드리아및마이크로솜분획의 COMT 활성도측정은효소시료와함께 3, 4-dihydroxybenzoic acid와 [methyl- 3 H] S-adenosyl-L-methionine이함유된 S-(5'-adenosyl)- L-methionine iodide를기질로사용하여 37 에서 30분간반응시키는동안에생성된방사성 3-hydroxy-4-methoxybenzoic acid와 4-hydroxy-3-methoxybenzoic acid를 toluene-isoamylalcohol (7 : 3) 혼합액으로추출한후그방사능을측정하여효소의활성도를산출하는 Borchardt (1981) 의법에준하였으며, 효소활성도단위는 1분간에 1 mg의단백질이반응하여생성한 3-hydroxy-4-methoxybenzoic acid와 4-hydroxy-3-methoxybenzoic acid의총량을 pmol로나타내었다. 이실험에서채택한효소활성도측정법들의정확도를높이기위하여같은시료에대하여 2회측정하여그평균치를취하였다. 이실험에서사용한방사능계측기는 Packard Tricarb 4530, liquid scintillation spectrometer (Packard사, 미국 ) 였다. 6. Km값및 Vmax 값의측정수술후 2일경과한모든실험군의세포분획효소시료들과 COMT의 2종기질중 3, 4-dihydroxybenzoic acid를선택하여이기질의원액과희석액을제조한후이기질액들과 [methyl- 3 H] S-adenosyl-L-methionine이함유된 S-(5'-adenosyl)- L-methionine iodide 원액을사용하여 COMT의활성도를측정한후이들성적으로부터 1/vi값을그리고기질농도로부터 1/[S] 값을계산하여이중역수도 (double reciprocal plot) 를그린다음이것으로부터 Km값과 Vmax값을산출하였다

3 7. 단백질정량효소시료중의단백질정량은 0.5 M perchloric acid와 methanol-ether 혼합액 (3 : 1) 으로단백질을정제하는 Greenberg et al. (1957) 법으로효소시료중의단백질을정제한다음 biuret법 (Gornall et al., 1949) 으로정량하였다. 8. 성적검정유의성검정은 Student's t-test로하였으며유의수준은 0.05 이하로하였다. 결과 1. 쥐에서간엽절제후재생간에서의 COMT 활성도변동간엽절제후재생간의세포질및마이크로솜 COMT의활성도는간엽절제후 2일및 3일에통계학적으로유의한증가를나타내었다. 즉간엽절제후재생간의세포질 COMT 의활성도는간엽절제후 2일에는원래간보다약 45% (P< 0.01), 3일에는원래간보다약 39% (P<0.0.5) 의증가를나타내 었으며 (Table 1), 재생간의마이크로솜 COMT의활성도는간엽절제후 2일에는원래간보다 47% (P<0.01), 3일에는원래간보다약 41% (P<0.01) 의증가를나타내었다 (Table 3). 그러나간엽절제후재생간의미토콘드리아 COMT의활성도는실험전기간동안통계학적으로유의한변동은나타내지않았다 (Table 2). Table 2. Activity of mitochondrial catechol-o-methyltransferase ±18 108± ±22 123± ±20 136± ±19 124± ±18 106± ±17 98±18 The data are expressed as mean ± SD with 5 rats in each group Table 1. Activity of cytosolic catechol-o-methyltransferase 0.5 2,876±452 2,825± ,892±467 3,314± ,910±456 4,208±652 b 3 2,918±454 4,045±615 a 6 2,894±448 2,886± ,872±443 2,866±448 The data are expressed as mean ± SD with 5 rats in each group. Significant difference from original liver; a, P<0.05; b, P<0.01 Table 3. Activity of microsomal catechol-o-methyltransferase ±62 436± ±68 496± ±65 632±87 b 3 428±70 605±94 b 6 424±66 432± ±64 423±67 The data are expressed as mean ± SD with 5 rats in each group. Significant difference from original liver; b, P<0.01 Table 4. Catechol-O-methyltransferase (COMT) kinetic parameters from regenerating rat livers determined with 3, 4-dihydroxybenzoic acid as substrate Cell fractions Vmax Km (mm) Original live Regeneration liver Original live Regeneration liver Cytosol 2.84± ±0.62 4,895±720 6,753±986 b Microsome 3.28± ± ± 95 1,057±136 b Michaelis-Menten constants for COMT were determined using 3, 4-dihydroxybenzoic acid, S-(5'-adenosyl)-L-methionine iodide and [methyl- 3 H] S-adenosyl-L-methionine at 37 for cytosolic and microsomal fractions of original, and regenerating rat livers at two s after partial hepatectomy. The data are expressed as mean ± SD with 5 rats in each group. Significant difference from original livers; b, P<

4 2. 간엽절제후 2일의쥐재생간에서 COMT 의 Km값및 Vmax 값의변동간엽절제후 2일의재생간에서세포질및마이크로솜 COMT의 Km값은변동이없었다. 그러나이들분획에서이효소의 Vmax값은모두통계학적으로유의한증가를나타내었다. 즉간엽절제후 2일경과시킨재생간에서세포질 COMT의 Vmax값은원래간보다약 38% (P<0.01) 의증가를나타내었으며, 마이크로솜 COMT의 Vmax값은원래간보다약 46% (P<0.01) 의증가를나타내었다 (Table 4). 고찰쥐에서간의중엽과좌측외엽을절제하면남은간엽은급격히재생되어비대해지며이때간조직은재생을위해핵산과단백질합성이활발해진다 (Lieberman et al., 1965). 또한이와같은간의재생기에는각종물질대사를촉매하는효소들의활성도도변동된다. 간재생이활발한시기의재생간에서그활성도가변동되는생체이물생체변환효소들은많으며그중에서도활성도가증가되는생체이물생체변환효소들은 monoamine oxidase, alcohol dehydrogenase, aldehyde dehydrogenase, microsomal ethanol oxidizing system, aryl sulfotransferase, arylamine N-methyltransferase 및 thiol methyltransferase이며 (Mun et al., 1988; Joo et al., 2002) 그활성도가감소되는생체이물생체변환효소들은 glutathione S-transferase, glutathione peroxidase, xanthine oxidase, superoxide dismutase, rhodanese, arylesterase, carboxylesterase 및 cholinesterase 등을들수있다 (Kwak et al., 1989; Joo et al., 2002). 이와같이간세포에존재하는생체이물생체변환효소들은간의재생기에간조직에서그활성도가변동된다. 따라서이실험에서측정한 COMT도간세포에주로존재하는만큼간의재생기에간에서그활성도가변동될수있을것이다. 이실험에서쥐의간엽을절제한후재생간의세포질및마이크로솜의 COMT 활성도는다같이간엽절제후 2일및 3일에유의한증가를나타내었다. 이성적으로보아 COMT 는간재생이왕성한시기에그활성도가증가되는효소라는것을알수있었다. 이실험에서간엽절제후 2일째의쥐재생간에서세포질및마이크로솜 COMT의 Km값과 Vmax값을측정했을때 Km값은모두변동이없었으나 Vmax값은모두유의한증가를나타내었다. 이와같이재생간에서이들효소의 Km값이변동이없으면서도 Vmax값이증가되고또한그활성도가증가된것은촉매효율증가에기인한것이라볼수는없다. 따라서재생간에서이효소의활성도가증가된것은그합성이증가되어나타난결과가아닌가생각된다. 재생기의간에서는간조직의재생을위하여우선적으로핵산과단백질합성이증가되고 (Lieberman et al., 1965) 아울러열량소대사도활발해진다 (Schofield et al., 1987; Dixit et al., 1992) 고한다. 이러한현상과함께간재생을위해유리한쪽으로먼저대사가진행되는것이라는가설 (Mun et al., 1988; Kwak et al., 1989) 이있어, 이실험에서측정한 COMT 는간재생과는유관한효소가아닌가생각된다. 그러나이실험만으로는재생간에서이효소의활성도증가가어떤원인에의한것인지는분명치않다. 따라서재생간에서이효소활성도의증가원인과기전을분명히알기위해서는앞으로더욱추구해보아야하겠다. 이상문헌상의지견과이실험결과를볼때쥐간의 COMT는간재생이활발한시기의재생간에서는그합성이증가되는효소로생각된다. REFERENCES Borchardt RT. Catechol-O-methyltransferase in Method in Enzymology (Jakoby WB. Ed) Vol 77, pp Academic Borchardt RT. N- and O-methylation in Enzymatic basis of detoxication (Jakoby WB. Ed) Vol II, pp Academic Dixit A, Baquer NZ, Rao AR. Inhibition of key enzymes of carbohydrate metabolism in regeneration mouse liver by ascorbic acid. Biochem Int : Gornall AG, Bardawill CJ, David MM. Determination of serum protein by means of biuret reaction. J Biol Chem : Greenberg DM, Rothstein M. Method for isolation and degradation of labelled compounds. in Method in enzymology (Colowick SP, Kaplan NO. Eds) Vol 4, pp Academic Joo I, Mun KC, Kwak CS. Thiol methyltransferase activity in regenerating liver after partial hepatectomy in rats. Keimyung Med J : Kim BK: Enzyme nomenclature, IUB pp Academic Kwak CS, Kim YH, Mun KC, Lee SH. Glutathione S-transferase and glutathione reductase activities in regenerating rat liver. Keimyung Univ Med J : Kwak CS, Kwak JS. Cell fractionation method of the rat liver. 1. Isolations of mitochondria and microsome. Keimyung Univ Med J : Lieberman I, Kane P. Synthesis of ribosome in the liver after

5 partial hepatectomy. J Biol Chem : Matsumoto K, Nakamura T. Molecular structure and function of hepatocyte growth factor. Metabolism (Jpn) : Mun KC. Catechol-O-methyltransferase activity in cholestatic rat's liver induced by bile duct ligation. J Biochem Mol Biol : Mun KC, Park EM, Kim YH, Kwak CS. Monoamine oxidase activity in regenerating rat liver. Keimyung Univ Med J : Raxworthy MJ, Gulliver PA, Hughes PJ. The cellular location of catechol-o-methyltransferase in rat liver. Naunyn-Schmiedeberg's Arch Pharmacol : Schofield PS, Sugden MC, Corstorphine CG, Zammit VA. Altered interactions between lipogenesis and fatty acid oxidation in regenerating rat liver. Biochem J :

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Microsoft Word - 09-곽춘식.doc J. Exp. Biomed. Sci. 13 (2007) 135 140 Effects of Intravenous Administration of Taurocholic Acid on Hepatic Catechol-O-Methyltransferase Activity in Rats with Choledocho-Caval Shunt Jun-Young Do 1, Kyo-Cheol