食餌性 Vitamin C의 吸收利用에 對한 硏究

柳總根,朱翼淳,金洙慶 中央醫學社 1967 中央醫學 Vol.12 No.4

To observe the absorbability of the dietary V-C in the body, the study was carried on the V-C concentration in the blood and urine after meal. The subjects, two of the nineteen years old boys were fed spinach, pickled radishes, tomato or vitamin C powder which were contained as much 200 mg as V-C respectively. The results obtained are summarized as follows, 1) Total V-C concentration in the blood showed the highest value at 3-4 hours after meal. 2) The highest V-C value in the blood was 1.89+0.13 mg/dl and it was in the saturated condition where as the total V-C excretion in the urine was about 47mg/day. 3) It seemed that the gut has a limitationn on the V-C absorption when a large amount of V-C was ingested.

食餌條件이 白鼠 腦의 Acetylcholine 및 Cholinesterase에 미치는 影響

류총근(Chong Kun Ryu),김용진(Yong Jin Kim),윤도선(Duo Sun Yun),임규태(Geu Tea Lim) 韓國營養學會 1973 Journal of Nutrition and Health Vol.6 No.1

소의 뇌에서 정제한 protein farnesyl cysteine carboxyl methyltransferase의 성질 분석

이안나,류총근,한종설,박길홍 고려대학교 의과대학 2000 고려대 의대 잡지 Vol.36 No.1

C-terminal farnesyl cysteine carboxyl methylation has been known to be the last step in the post-translational modification processes of several important signal transduction proteins in eukaryotes, mainly G-protein superfamily including ras related GTP binding proteins and the γ-subunit of heterotrimeric G proteins. Thus, regulatory role of protein farnesyl cysteine carboxyl methyltransferase (PFC- CMT; EC, 2.1.1.100) catalyzing the reaction is suggested for various physiologic activities of G proteins. And the enzyme was well characterized to be stimulated by guanosine 5'-O- (3-thiotriphosphatei (GTPγS) and suppressed by N-acetyl-S-farnesyl-L-cysteine (AFC). As an initial step to understand the physiological significance of the process, we attempted to purify the enzyme. The enzyme was partially purified 130-fold (specific activity, 143 pmol of methyl group transferred/ min/㎎ of protein) with an yield of 1.8% after the purification by fast protein liquid chromatography(FPLC) on Superdex 75 column, and further purified with non denaturing polyacrylamide gel electrophoresis (ND-PAGE) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-RAGE). The molecular weight of PFC- CMT was determined to be about 30 kDa based on Superdex 75 FPLC as well as photoaffinity labelling with S-adenosyl-L-〔methyl-³H 〕 methionine(〔methyl-³H 〕SAM). The partially purified enzyme(Superdex 75 eluate) was found to he characteristically stimulated by GTPγS. being about 40-fold increase of the activity in the presence of 2mM, in contrast to ATP which did not have any effect on the enzyme activity. Meanwhile, the enzyme was found to be markedly inhibited by AFC, reaching 0 activity- in 2mM, which strongly suggested that the partially purified enzyme was PFCCMT. 30 kDa protein extracted from SDS-PAGE was subjected to amino acid analysis and protein internal microsequencing by in gel digestion. The protein is composed of 266 amino acids, with leucine(l4 mole %) and alanine(l3 mole %) as major amino acids. Elution pattern of tryptic digests of the protein run on a capillary HPLC/ion trap mass spectrometer(LCQ) system showed no match in OWL database using the SeQuest program. Partial amino acid sequence of the protein, Leu-Gln-Leu-Arg and His-Pro-Val-Gly-Val-Glu-Tyr- Ala-Thr-Lys were obtained by Edman sequencing of the tryptic digests. The first sequence was too short to search, but there was no match for the second sequence in the NCBI nr or Est databases.

人蔘脂溶牲 成分이 四鹽化炭素 投與 白鼠의 蛋白質代謝에 미치는 影響

朴鍾培,柳總根 고려대학교 의과대학 1984 고려대 의대 잡지 Vol.21 No.1

In order to observe a part of the effect of Ginseng fat soluble components (Gx) on the protein metabolism under CCI₄ poisoning, 192 male and female albino rats were taken as subjects which were divided into 6 experimental groups i.e. control grop; CCI₄ group which were observed after CCI₄ and been administered only one time; Gx group which were observed administering Ginseng one time daily; CCI₄+Gx group which were observed administering Ginseng one time daily after CCI₄ had been administered, Gx+CCI₄ group to which CCI₄ was administered only on time after Ginseng had been administered 6 times; and Gx_CCI₄+Gx group to which Ginseng was administered one time daily after CCI₄ had been administered following a 6th time administration of Ginseng. Programed administrations of Ginseng or CCI₄ were given to each corresponding groups at several periods i.e. 1,2,4,6 and 8 days after the intial administration of given doses, changes of total protein, albumin, globulin, urea nitrogen, transaminase (GOT) of serum and total nitrogen observed at each period were measured and the results obtained are as follows: 1. Gx administration was found to have the ability to decrease the elevated SGOT activity resulting from administration of CCI₄ and induce rapid recrovery from such an elevated level and the extent of such a dereasing action was greatest in Gx+CCI₄+Gx group, than Gx+CCI₄ group, and the least in CCI₄+Gx group. 2. Gx+CCI₄+Gx group showed nearly no change in serum albumin level, and being in second place, Gx administering groups (CCI₄+Gx group, Gx+CCI₄ group) showed small change. 3. Gx administering groups showed more increase in globulin level at 1-2 day period than the CCI₄ group and during the same period A/G ratio was found inverted. 4. Total nitrogen amount in the liver was remarkably decreased in Gx administering groups at 2 day period. 5. Urea nitrogen was increased in CCI₄ group at 2 day period, whereas Gx administering groups showed decreases. 6. Great changes in electrophoresis fractions were found in α₁-and r-globulin fractions at 1-2 day period. For more detail a. α₁-globulin; Gx administering groups showed lower increases than the CCI₄ group at 2 day period. b. r-globulin; Gx_CCI₄ group and CCI₄+Gx group showed more increases than the CCI₄ group at 1-2 day period. 7. α₂-globulin, revealing characteristic pattern, was increased to the same extent in all the Gx administerings groups during Gx administering period, thus Gx was estimated to increase the α₂-globulin component.

Immunoadsorbent에 의한 γ-Glutamyltransferase Isozyme의 硏究

金美惠,柳總根 고려대학교 의과대학 1989 고려대 의대 잡지 Vol.26 No.3

The purpose of this study is to prepare immunoadsorbent for the purification of γ-Gluta-myltransferase(γ-GT) with which the purification course is simple and purification fold is increased. For purifying γ-GT used as antigen to develop immunoadsorbents, normal rat liver was treated with papain, fractionated with ammonium sulfate(45-90%), which was followed by DE-52, Con A-Sepharose, Sephadex G-200 chromatography. Isozumes were purified from rat liver, kindey, and hepatoma mass with imminoadsorbent to investigate the isozyme patten on Con A-Sepharose chromatography and polyacrylamide gel electrophoresis, and their Km values. The following results were obtained. 1. By immunoadsorbent, γ-GT were purified 3397, 498, and 1030 fold with an yield of 12,13, and 17%, and the final specific acticities were 10.6,597, and 30.9 units per mg of protein, from liver and kidney of normal rat, and hepatoma, respectively. 2. On Con A-Sepharose chromatography, sialo γ-GTs from normal rat liver and hepatoma accounted for 5.1% and 28% of the total γ-GT, respectively. On the other gand, immunoadsorbent could bind both asialo and sialo γ-GT. 3. There were no differences in Km values for γ-Glitamy1-ρ-nitroanilide regardless of the purification methods, that is, immunoaffinity chromatography or the previous mrthod employing papain treatment and ammonium sulfate fractionation followed by DE-52, Con A-Sepharose, and Sephadex G-200 chtomatography in case of all the enzyme sources involving liver and kidney of normal rat, and hepatoma mass. 4. On 5-20% gradient polyacrylamide gel electrophoresis, the frations γ-GTs from the same tissue were located in the same positions, in spite of different purification methods. The extent of purification was higher in purification with immunoaffinity chromatography than that with the previous method. 5. From the results described above, purification with immunoaffinity chromatography needed preparing anti-γ-GT antibody, but increased purification fold and simple purification course could be obtained.

정상 난소 및 난소암조직에서 20 kDa 단백질의 arginine 메칠화 반응에 관한 연구

이교원,류총근,한종설,박길홍 고려대학교 의과대학 2000 고려대 의대 잡지 Vol.36 No.1

A1 protein methylase I(A1 PMI) activity has been well known to increase in highly proliferative cells, intracellular substrate of which, 20 kDa protein, has been discovered recently. Present study intended to investigate the relevance of ovarian cancer development and arginine methylation of the protein, comparing the intensities of 20 kDa protein and A1 protein arginine methylation in normal ovary and ovarian cancer tissues. Normal ovary and ovarian cancer tissues were taken from the same ovarian cancer patient. A1 protein expression was induced 30-40 fold by IPTG in BL21(DES3)LysS E-coli, and A1 protein was purified to apparent homogeneity with an yield of 2.89㎎ per g E-coli. Specific activities of A1 PMI in normal and cancer ovarian tissues were 0.17, 0.2, 1.18 fold higher in cancer tissue for intracellular substrates, and 5.6, 7.0. 1.25 fold higher in cancer tissue for A1 protein. Fluorography revealed methylated 20 kDa protein as an only intracellular substrate of A1 PMI, the intensities of which for normal and cancer ovarian tissues corresponded to enzyme activity measurements. When A1 protein was added as exogenous substrates, methylated A1 was observed, which parelled the enzyme activities in applied samples, and 20 kDa protein was found to disappear. Conclusively, it was confirmed that 20 kDa protein is the most favored intracellular substrate for AlPMI in physiologic condition. and the protein and A1 protein are methylated by the same enzyme competitively.

단일클론 항γ-Glutamyltransferase항체를 이용한 효소 및 방사 면역측정법 적용에 관한 연구

金明坤,柳總根 고려대학교 의과대학 1994 고려대 의대 잡지 Vol.31 No.1

γ-Glutamyltransferase (GGT: E. C. 2. 3. 2. 2.) is a glycoprotein enzyme which is involved in glutathione metabolism and amino acid transport through the plasma membrane. It is distributed widely in several organs including liver and kidney. Several isozymes of GGT have been reported and some of the isozymes may be associated with hepatocarcinogenesis. We have produced six monoclnal antibodies (mAbs) against GGT purified from the liver of 2-acetamidofluorene (AAF) treated rats. All of the six mAbs were obtained by immunizing mice with liver GGT. Liver GGT from AAF treated rats was biochemically purified to a specific activity of 125.6 units per mg of protein. The overall purification was 551 folds and the final yield was 20. 8. Six hybridomas which produced anti-GGT Abs were extensively subcloned and injected into the peritoneal cavity of BALB/c mice to obtain large quantities of Abs. These mAbs were purified from ascites by ammonium sulfate precipitation and protein A sepharose CL-4B column chromatography. Using these mAbs we performed enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunohistochemistry (IHC), and autoradiography (ARG) to study the distribution of GGT isozymes in tissue. The results indicate that GGT-mAb 1 is specific for the AAF treated liver GGT, GGT-mAb 5 for the normal liver GGT, and GGT-mAb 6 for the normal kidney GGT. These mAbs may be used to evaluate the distribution of GGT isozymes in different tissues.

白米와 麥類混食에 衣한 白鼠體內 蛋白質代謝에 關한 硏究

文翰培,柳總根 고려대학교 의과대학 1974 고려대 의대 잡지 Vol.11 No.1

This study was carried out to observe the effect of individual species or mixed ratio of wheat and barley with rice diet on the metabolic change of protein in growing male albino rat. The species of wheat and barley used were wheat, naked barley (Baik-dong and Cheoung-maik), and barley (Su-won No. 18 and Jei-cheun No. 5). The growing rats were fed on 17 kinds of the mixed diets, such as 10%, 30% and 50% wheat or barley mixed with rice diets for 12weeks, respectively. The contents of hemoglobin and hematocrit in the blood, and total nitrogen, amino acid nitrogen, urea-nitrogen, and creatinine in the liver and serum, and urea-nitrogen, creatinine, and urea- nitrogen/creatinine ratio in the urine have been measured. The results obtained are summarized as follows: 1. The contents of the hemoglobin and the hematocrit were no remarkable difference in between each mixed diet group compared with the control group. 2. The total nitrogen contents in the liver and the serum of the wheat or barley mixed diet group were higher than that of the standard diet group, but these were the similar to the control diet group. 3. The amino acid nitrogen contents in the liver and the serum of the mixed diet groups showed a tendency to decrease but these were higher or similar than that of the control diet group. increase after four weeks feeding. 4. The creatinine contents in the liver of the control and each diet group showed a tendency to increase after four weeks feeding. 5. In the urine, the urea-nitrogen and creatinine contents were decreased after four weeks feeding and these were excreted very small amounts by feeding the control and each diet group for 12 weeks. 6. The urea-nitrogen/creatinine ratio in the urine of all diet group were decreased gradually after four weeks feeding. It is concluded form the above results that the contents of amino acid nitrogen in the liver and the serum, and urea-nitrogen, creatinine, and urea-nitrogen/creatinine ratio in the urine of all diet groups were decreased after four weeks feeding. But the protein metabolism was not greatly affected by the species or mixed ratio of the wheat or barley mixed with rice in the growing albino rat.

白鼠 腸管內 遊離아미노酸의 變動에 關한 硏究

朱軫淳,柳總根 고려대학교 의과대학 1976 고려대 의대 잡지 Vol.13 No.2

This study was carried out to observe the changes on metabolism of endogenous protein in animal which was maintained with protein free diet. The animal, 72 adult male albino rats, 350g-390g of body weight, were used for the experiments, and they were divided into two diet groups, the control and the protein free diet groups, and the latter group was again divided into 7 groups depend on feeding term: 2, 4, 7, 14, 24, 42 and 62-day-group. The changes of the body weight were observed for 62 days and the values of total nitrogen and the free amino acid contents in the serum and the gut content were determined by the method of micro Kjeldahl and gas-liquid chromatography respectively. The results obtained are summerized as follows. 1. The body weights were decreased gradually through the feeding periods of protein free diet, then decreased 42% to 46% of initial body weight during 62 days feeding of the diet. 2. The values of total nitrogen in the serum and the gut contents were similar to that of the control group during feeding of protein free diet for 24 days, then that of the serum was maintaintained t o almost constant levels until 62-day, but that of the gut content was increased in 24-day to 62-day. 3. The changes of free amino acid contents: a) The values of the total free amino acid in the serum were higher than the control group until 14 days feeding of the protein free diet, but there-after the values were decreased slowly, then 62-day-group have been lower value than the control group. Whereas, the values of total amino acid in the gut content were lower than that of the control group until 7 days feeding, after 42 days feeding, the values were shown a increased tendency. b) The values of isoleusine and leucine in serum were similar to that of the control group for 42 days feeding of the protein free diet, but the values were lower than the control group in 62-day-group. While, the both values in the gut content were lower than the control group for 14 days feeding, but there-after the values were increased to higher values than control group. c) The value of lysine in the serum was slightly lower than the control group for 24 days feeding of the protein free diet, but the value was maintained to similar level with the control group in 42-day-group. While, the value of lysine in the gut content was shown similar tendency with the change in serum except after 42-days d) The changes of methionine values in serum and gut content were contrary to each other throughout the period. e) The values of phenylalanine in serum and gut content were kept to similar value of the control group until 42 days feeding. f) Threonine value in serium was decreased continuously for all feeding period, then the value of 62-day-group was become to half value of the control group' While the value of the gut content was similar to value of the control group until 24 days feeding, there-after the value was increased. g) The values of tryptophan in serum and gut content were trace level during 42 to 62 days feeding of protein free diet. h) The balues of alanine, glutamic acid, glycine, serine and proline in serum were significantly higher than that of control group during 14 to 24 days feeding of protein free diet, but the values of 42-day-group were decreased to levels of the control group. In view of above results, it would be empasized that the ability of metabolic homeostasis was relatively well behaved for 7~14 days, there-after, the ability was decreased by feeding of protein free diet, and the ability in gut homeostasis was contrary to metabolic homeostasis.

암세포의 증식과정에서 20 kDa 단백질의 arginine 메칠화 반응에 관한 연구

최석민,류총근,한종설,박길홍 고려대학교 의과대학 2000 고려대 의대 잡지 Vol.36 No.1

Enzymatic methylation of endogenous protein (s) by protein arginine N-methyl- transferase(EC. 2.1.1.23) in several cancer cell lines was investigated in an attempt to understand possible regulatory role of the modification in relation to cellular proliferation. When several cancer cells (HeLa, HCT-48, A549, HepG2) were incubated with S-adenosyl-L-〔rnethyl-³H〕methionine, the endogenous 20- kDa species in the extracts were most intensely 〔methyl-³H 〕-labeled compared to that in normal colon tissues. Whether the 〔methyl-³H 〕-labeld 20-kDa is due to carboxyl-O-methylation, the alkali-lability of the purified and methylated 20- kDa species were examined at pH 9-11, at 37℃ for 60 min. The results indicated that 〔methyl-³H 〕-label on the 20-kDa species of HCT-48 cells did not decrease after alkali treatment quantified by Fuji BAS 2500 Bio-Image analyzer. The methylation of 20-kDa did not increase in the presence of 4 mM GTPγS, further indicating the methylation is not due to C-terminal farnesylated cysteine carboxyl methylation. Also, none of purified histones showed the same mobility on SDS-PAGE as the 20-kDa endogenoiis substrate. In conclusion, in the present study, a new novel endogenous 20-kDa methyl accepting protein for protein arginine methyltransferase was found which is closely correlated with cell proliferation suggesting a possible role of this post-translational modification during cellular proliferation.