胎盤組織의 Nucleases에 關한 硏究(Ⅱ) : 사람 胎盤組織의 Alkaline Deoxyribonucleases의 性狀 Properties of Alkaline Deoxyribonucleases from the Early Human Placenta

白汶基,黃炳斗,李正馥,琴基昌 충남대학교 의과대학 지역사회의학연구소 1978 충남의대잡지 Vol.5 No.1

Properties of alkaline deoxyribonucleases from cytosol and membane fractions of the early human placenta have been investigated. Evidence is indicating that the cytosol deoxyribonuclease (DNase) having an optimal pH around 9 and the plasma membrane bound DNases having an optimal pH around 8 (pH- 8-PM-DNase) and 9. 5 (pH-9.5-PM-DNase), respectively, are distinct enzyme entities. The cytosol DNase, partially purified, is activated by 1mM CO^2+ and the pH-8-PMDNase and the pH-9. 5-PM-DNase are required Mg^2+ and Co2^2+, respectively, for its maximal activity. The cytosol DNase is relatively heat-labile, whereas the plasma membrane bound DNases are heat-labile with some differnce. The above DNases act both on the native DNA and the heat-denatured DNA with preference for the latter. Kinetic analysis shows that the maximal velocity is independent of Co^2+, while Km decreases from 4. 16 × 10 exp(-4) M DNA-p in the absence of Cot^2+ to 9. 4×10 exp(-5) M DNA-p in the presence of Co^2+. From the above result and the fact that the DNA is not only associated with membrane structure, but also membrane-DNA interaction may be relevant to several pathologic process, including aging and carcinogenesis, we discussed that the PH-9-PM-DNase is related to the differenciation of the early placenta.

胎盤組織 S-Adenosyl-L-Methionine : Protein-Carboxyl-O-Methyltransferase에 關한 硏究 Protein-Carboxyl-O-Methyltransferase from Human Placenta

黃炳斗,白汶基 충남대학교 의과대학 지역사회의학연구소 1983 충남의대잡지 Vol.10 No.2

S-Adenosy-L-methionine: protein-carboxyl O-methyltransferase (protein methylase Ⅱ) has been purified from human term placenta approximately 8,700-fold with a 14% yield. the enzyme shows a sharp pH optimum around pH 5.8. The enzyme is easily inactivated by heat treatment for 5 minutes at 60℃, and when stored at -20℃ even in the presence of 10% glycerol, approximately 70% of activity has been lost in 8 weeks. Copper ion (Cu^2+) is a potent inhibitor, the activity being completely inhibited at 2 mM and 86% of the activity is recovered by addition of 4mM EDTA. The other divalent cations are almost without effect. Human chorionic gonadotropin, immunoglobulin A, and histone ⅡA are good substrates for this enzyme with preference for HCG. The Km value for S-adenosyl-L-methionine and Ki value for S-adenosyl-L-homocysteine are 2.08 × 10 exp (-6) M and 5.8 × 10 exp (-7) M, respectively. The methylated membrane protein from human placenta are analyzed by dodecyl sulfatepolyacrylamide gel electrophoresis. The bands 9 and 17 of the membrane protein are identified as two major classes of methyl acceptors, but in the presence of epinephrine, methylation of the band 9 is decreased and the bands 13, 14, 16 and 17 become major methyl-acceptors. Especially methylation of the band 17 is increased about 3-fold compared with that of the band 17 in the absence of epinephrine. The nuclear membrane proteins are found to be a good methyl-acceptor protein. The enzyme activity with nuclear fraction as substrate is increased about 17% when 39,000 x g supernatant and prednisolone are added together to reaction mixture. Methylated histone prefers to bind methylated nucleus rather than nonmethylated nucleus. The enzyme activity is changed with gestational age, the activity at first and second trimester being about 2 times higher than that of term placenta. From the above results, the possible roles of this enzyme, such as a regulation of hormone action, transport of histone and steroid hormone into the nucleus and signal transmission, are discussed.

胎盤組織의 Adenosine Deaminase

황병두,이재흔,백문기 충남대학교 의과대학 지역사회의학연구소 1981 충남의대잡지 Vol.8 No.2

Adenosine deaminase(ADase) has been partially purified approximately 800-fold from human placenta, and its properties have been investigated. The enzyme exhibits a broad pH optimum from pH 6 to 8 and is heat-labile, being completely inactivated by heat treatment at 70℃ for 10 minutes. In contrast to other tissue ADase, the enzyme is inhibited by 2mM of Cu^2+ and Fe^2+ at pH 7, and by 2 mM of Cu^2+, Fe^2+, Co^2+ and Mn^2+ at pH 8. The inhibitory effect of Cu^2+ on the enzyme is so strong that the enzyme is completely inactivated by 20 μM of Cu^2+. The enzyme activity is easily recovered by EDTA(4 mM) in case of adding Cu^2+ after preincubation of enzyme with substrate, while the recovery is about 50% by treating Cu^2+ prior to addition of substrate. Thus substrate binding to enzyme seems to prohibit the Cu^2+ from binding to the enzyme. The apparant Michaelis constant for adenosine is 77μM, and the kinetic analysis in the presence of 2 μM Cu^2+ shows that Km is independent while Vmax is decreased. The molecular weight of the enzyme is about 800,000 and the apparant activity of the other type of ADase is not detected. From the above results it is suggested that the enzyme is different from other tissue ADase in its nature of protein and possibly biologic role.

胎盤 組織의 Protein Methylase II

황병두,이재흔,백문기 충남대학교 의과대학 지역사회의학연구소 1982 충남의대잡지 Vol.9 No.2

Protein methylase Ⅱ has been purified from human term placenta approximately 8,760 folds with a 14.5% yield. The enzyme shows sharp pH optimum around pH 5.8. The enzyme is easily inactivated by heat treatment for 5 minutes at 60℃, and when stored at -20℃ even in the presence of 10% glycerol, approximately 70% of activity has been lost for 8 weeks The enzyme doesn't require any divalent cations and Cu^2+ is a potent inhibitor, the activity being completely inhibited at 2 mM and 86% of the activity is recovered by addition of 4 mM EDTA. Histone IIA and myelin. basic protein arc good substrate for this enzyme. Km for S-adenosyl-L-methicnine and Ki for S-adenosyl-L-homocysteine are 2.03 x 10 exp(-6) M and 5. 8 x 10 exp(-7) M respectively. The methylated membrane protein from human placenta were analyzed by dodecyl sulfate/polyacrylamide gel electrophoresis. Band 1, 2, 4, 15, 17 are identified as 5 major classes of methyl-acceptor protein for protein methylase Ⅱ. The role of placental membrane prctein methylation is discussed with regard to placental function.

精神分裂症의 生物學的 硏究 : 液體 크로마토그라프와 放財酵素法에 依한 Catecholamines 微量定量 A Rapid Microassay of Catecholamines by High Performance Liquid Chromatography-Radioenzymatic Method

고병학,송옥헌,임규,황병두,백문기 中央醫學社 1983 中央醫學 Vol.45 No.4

A direct rapid radioenzymatic assay in combination with high performance liquid chromatography (HPLC) for measuring simultaneously picogram quantities of nore pinephrine, epinephrine and dopamine has been developed, and the relationship of changes in cerebrospinal fluid (CSF) norepinephrine concentration during haloperidol treatment to changes in psychosis rating of twelve schizophrenic patients has been investigated. The three catecholamines in 20?l of CSF are converted to their 0-methylated ana-logues by catechol-0-methyltransferase in the presence of S-adenosyl-L-1{methyl-3H} methionine. After the addition of tetraphenylborate, these derivatives are extracted. with diethylether at pH 8.2 followed by re-extraction with 0.1N HCI and then isol-ated by HPLC using, ?Bondapak C18column and 0.17 M acetic acid-60% methanol- 0.02 M Na2SO4 as solvent. Manual collection of each fraction is started two seconds after in appeared mark. The HPLC fraction of norepinephrine and epinephrine deri-vatives are oxidized to vanillin followed by extracting the vanillin with toluene, and dopamine derivatives fraction is extracted with toluene/isoamylalcohol and then re-extracted with 0.1N HCl. Internal standard curve shows a good linearity from 5 to 30 picogram per samples, and incorporated radioactivity of norepinephrine, epinephrine and dopamine derivatives are 163±11.4, 125±10.5 and 112±3.9cpm per picogram and calculated sensitivities are 0.4, 0.3 and 1 picogram, respectively. 3H-Normetanephrine, 3H-metanephrine and 3H-methoxytytamine are well separated by HPLC, and each retention times are 3.52, 4.08 and 5.62 minutes and yields are 75.6?0.9, 76.1?0.8 and 70.1?0.4%. Separation of the methylated derivatives by HLPC are performed within 8 minutes, and thus enabled us to complete the differential analysis of 15 samples (30 tubes) in a day. CSF norepinephrine concentration in schizophrenic patients show rostral-caudal gradient and haloperidol induced decreases of norepinephrine and the decreases correlated significantly with psychosis rating, From the above rsults, it is suggested that this method is more excellent than any others in sensitivity, accuracy, reproducibility and specificity of the differential assay, and CSF norepinephrine can be used for evaluation of neuroleptics treatment of schizophrenia by this method.

初期 胎盤組織의 Adenosine Deaminase

황병두,변해공,임규,이재흔,백문기 충남대학교 의과대학 지역사회의학연구소 1983 충남의대잡지 Vol.10 No.2

Two forms of adenosine deaminase (ADase) having molecular weights of 1,500,000 & 800,000 in the soluble fraction of the early placenta and plasma membrane bound ADase of the placenta were demonstrated respectively. Their properties and activity changes with gestational age have been investigated. ADases exhibit a broad pH optimum from 6 to 9 and heat labile, being completely inactivated by heat treatment at 70℃ for 10 min. Soluble fraction ADases and plasma membrane bound ADase are inhibited about 100% and 80% by s mM Cu^2+. The apparent Michaelis constant of pADase-cytotrophoblast(pADase-C) for adenosine is 110μM, and for deoxyadenosine 21μM, that of pADase-syncytiotrophoblast(pADase-S) is 80μM and 35μM, and that of plasma membrane bound ADase is 31μM and 26μM, respectively. Kinetic analysis of pADase-C and pADase-S in the presence of 10μM Cu^2+ show that the nature of the inhibition to pADase-C and pADase-S are noncompetitive and uncompetitive, respectively. The pADases (pADase-C + pADase-S) activity decreased with gestational age, the activity at term being about one-tenth that of the early placenta, while plasma membrane bound ADase increased with gestational age by about 2-fold. From above results it is discussed that pADase-C is related to cytotrophoblast, pADase-S to syncytiotrophoblast and plasma membrane bound ADase to cellular differentiation of placenta, and suggested that soluble fraction ADases of human placenta is different from plasma membrane bound ADase in their biological roles.

胎盤組織 Cytidine Deaminase에 關한 硏究 (Ⅰ) : Purification & Characterization

황병두,곽상태,임규,이재흔,백문기 충남대학교 의과대학 지역사회의학연구소 1984 충남의대잡지 Vol.11 No.2

Cytidine deaminase has been partially purified from human term placenta approximately 26-fold by combination of ammoninm sulfate saturation, sephacryl S-300, DEAE-cellulose and hydroxyapatite chromatography, and then its properties have been investigated. The enzyme shows a broad optimum pH from 5 to 9 and is heat-stable being, almost remained its activity by heat treatment at 70℃ for 8 minutes. The enzyme activity is inhibited to 23% and 77% by 2 mM of Cu^2+ and Co^2+, respectively, but, other cations do not affect the enzyme activity. The apparant Michaelis constant for cytidine and deoxycytidine are 74 uM and 69 uM, respectively. Kinetic analysis of cytidine deminase in presence of 0.25 mM Cu^2+ shows that the nature of the inhibition to cytidine deaminase is competitive, considering that Vmax is independent while km value is increased. The enzyme activity is completely inhibited by 0. 5 mM of p-hydroxymecuribenzoicacid(PHMB)but all of the activity is recovered in adding mercaptoethanol. The enzyme activity is decreased about 50% in prescence of 5 mM of polyamines. The enzyme activity is mainly located in the nuclei (80%) and cytosol(16%) fractions. he molecular weight of the enzyme is about 770, 000.

사람 洋水의 Adenosine Diphosphoribose Pyrophosphohydrolase

黃炳斗,白汶基 충남대학교 의과대학 지역사회의학연구소 1975 충남의대잡지 Vol.2 No.2

Adenosine diphosphoribose pyrophosphohydrolase (ADPR-PPase) which catalyzes the hydrolysis at the pyrophosphate linkage of adenosine diphosphoribose (ADP-ribose) formed from nicotinamide adenine dinucleotide (NAD) by NAD nucleosidase has been partially purified from human amniotic fluid, and its properties have been investigated. This enzyme shows optimal pH around 9.5 and is heat-labile, being completely inactivated by heat treament at 65˚ for 10 minutes. In contrast to erythrocyte ADPR-PPase of rabbit, the enzyme does not require Mg^2+, but requires inoragnic phosphate for the activity, maximal activity being attained at 20mM phosphate. The apparent Michaelis constant for ADP-ribose is 1.4 mM. The enzyme is inhibited markedly by adenine nucleotides which act as a competitive inhibitor, although CMP and GMP are also inhibitory, yet lesser in magnitude, whereas oxidized and reduced NAD and pHMB are not inhibitory. These results indicate that the enzyme is controled by related compounds as well as reaction product.

植物凝集素 Terminalis chebula R.C. 및 토끼赤血球에 의한 사람 血淸型에 關한 硏究

張丙錡,文國鎭 고려대학교 의과대학 1985 고려대 의대 잡지 Vol.22 No.3

Plant extracts have variable biological activities, as phytagglutinins, mitogens, lymphocytotoxins, anticancer substances, sperm agglutinins and so on. So they have been used extensively in medical field, including medicolegal part. In medicolegal part, plant extracts, as phytagglutinins (PHA), were mainly used for classification of new human red blood cell types and for detection of new lectins up to date. But recently some investigators attempted to classify human sera by their inhibition properties on PHA. For the purpose of new classification of human serum, the author used Terminalis chebula R.C. extracts, which act as panphytagglutinin to rabbit red blood cells, by use of inhibition property of fresh human serum, blood stains, whole blood stains and various blood type to PHA. The results of this study could be summerized as follows; 1. Human sera could be classified into two groups. One was possessed of certain substance which inhibit the agglutination activity of Terminalis chebula R.C. extracts on rabbit RBC and the other was not. 2. The frequency of the former was 15% and the latter was 85% respectively. 3. The agglutination inhibition phanomena of Terminalis chebula R.C. extracts on rabbit RBC by human sera showed no relation with ABO, Cl, H, ab.C, CCI, PVI, Cea, WCea, and Hp blood types respectively. 4. This serum type may be available for classification of blood stains.

사람 胎盤組織의 Alkaline Deoxyribonuclease

白汶基,黃炳斗 충남대학교 의과대학 지역사회의학연구소 1976 충남의대잡지 Vol.3 No.2

Serial estimation of acid and alkaline deoxyribonucleases of human placenta has been performed, and some properties of alkaline deoxyribonuclease having an optimal pH around 9 has been investigated. A gradual decrease in the acid deoxyribonuclease activity occurs with advancing gestation after 20 weeks, the activity at term being about one fourth of that of before 20 weeks. The alkaline deoxyribonuclease activity in maintained until approximately the 16th week of gestation at which the functioning villi has been established and thereafter decreases rapidly with no apparent activity about 28 weeks. The alkaline deoxyribonuclease is heat-stable with 30% activity even at 90℃ for minutes. The alkaline deoxyribonuclease is inhibited by Ca^2+, Mn^2+ and ethylenediaminetetraacetate and is activated by Co^2+, Mg^2+, ATP and cyanocobalamin, the optimal concentration of Co^2+ necessary for maximal activity being 3 mM. The alkaline deoxyribonuclease acts both on the native DNA and heat-denatured DNA with preference for the latter. Kinetic analysis shows that the maximal velocity is independent of Co^2+ while Km decrease from 4.16 × 10^-4 M DNA-P in the absence of Co^2+ to 9.4 × 10^-5 DNA-P in the presence of Co^2+. From the activity changes with gestational age, it is suggested that the alkaline deoxyribonuclease plays an important role in the differentiation of the function villi.