Multidrug resistance-associated protein (MRP) and multidrug resistance (MDRI) gene expression in osteoarcoma and their prognostic significance

학위논문(박사)--서울대학교 대학원 :의학과 정형외과학전공,2000.Docto

정상보행 및 속보시에 족지 굴근이 족관절 족저굴곡력에 미치는 영향 : 3차원적 동작분석을 이용한 운동역학적 분석

학위논문(석사)--서울대학교 대학원 :의학과 정형외과학전공,1998.Maste

Mechanism for Increase in 5'-Nucleotidase Activity of Rat Liver Plasma Membrane npon Aging in vitro

5'-Nucleotidase is one of the marker enzymes for plasma membrane in subcellular fractionation. In recent years it ia sbown that the enzyme is not only important in its diagnostic value for hepatic meta' static carcillOma. but also responsible for its regulation of blood Sow through hydrolysis of AMP to adenosine. a powerful vasodilator. It was reported as well that its activity of plasma membrane is increased upon aging the membrane. While most enzymes bound to membranes decrease in their activities upon aging the membranes in vitro, 5'nucleotidase activity is increased to the contrary under the same condition. The present investigation was carried out to elucidate the possible mechanism involved in the phenomenon. Plasma membrane was purified from rat liver homogenate by sucrose density gradient and was suspended in isotonic sucrose solution. part of which was sonified and ultracentrifuged to obtain membrane- free supernatant, and both samples were used as enzyme sources. The behaviors of 5'-nucleotidase were observed in the control system consisting of substrate and buffer. and in the activation system consisting of substrate, buffer, sodium deoxycholate, and MnCI•. The results obtained are summarized as follows. 1. From the results showing the increased activities of 5' -nucleotidase of both the plasma membrane suspension and the membrane-free supernatant upon aging the samples, it is concluded that it is independent of the membrane structure but is due to the property of enzyme molecule itself. The 5'nucleotidase activities of both the suspension and the supernatant, measured in the activation system were approximately 100% and 70% respectively higher than those measured in the control system. After incubation of the suspension at 45°C for 1 hour. the 5' -nucleotidase activity was increased by 16% in the control system, while it showed no variation in the activation system. 2. The increase in 5'-nucletidase activity caused with deoxycholate in the absence of Mn* in the assay system was a very slow reaction but the reaction reached immediately to a maximum in the presence of Mn*. 3. Apparent Km values for 5' -riucleotidase measured with the same amount of suspension In the control system and in the activation system were the same, O. 12mM but Vmax for the reaction was about 2-fold greater in the activation system than that measured in the control system. 4. The increase in 5'-nucleotidase activities of the supension and the supernatant was directly dependent on the concentration of deoxycholate in the presence of Mn* in the assay system, while their rates of increase upon aging the enzyme preparation were inversely dependent on the concentration of deoxycholate. 5. The above results may well be explained reasonably by assuming the presence of a regulatory substance. R being associated with the catalytic enzyme molecule, C of 5'-nucleotidase; C, an active form of the enzyme, whereas C.R, a latent form of the enzyme. From the foregoing results it can be inferred that C could be a thermally stable protein, whereas R may be a thermally unstable substance; the latter being slowly denatured upon aging and released from C' R, which leads eventually to the increase in active form. It can also be inferred that R is released from C·R by deoxycholate in the activation system for which the following equdtion could be suggested, Mn* C·R+D=C+R·D(D, the molecule of deoxycholate

A Study on Staining, Determination and Regulatory Mechanism of Guanine Deaminasc

For preparation of the isoenzymic forms of guanine deaminase(GDA). the supernatant from rat liver homogenate was subjected to salting-out, followed by dialysis and DEAE-cellulose column chromatography. The fractions collected from the column were pooled into Franction A, Band C according to the chromatographic elution pattern. To characterize the isoenzymic nature of each fraction, the enzyme kinetic properties were studied and the isoenzymcs were visualized on the polyacrylamide gel after electrophoresis. The results obtained are summarized as follows: I. GDA isoenzymes were stained by coupling with a flavoproetein, xanthine oxidase in the presence of an elecron carrier, phenazinc methosul£atc and nitro blue tetrazolium which in turn is reduced to a water-insoluble dye, formazan. 2. Fraction A was the first-eluted peak of GDA activities from the DEAE-cellulose column and was separated into two major bands on the polyacrylamide gel after electrophoresis which showed slow migration through the gel. The slowest-moving band of Fraction A was not shown in the electropherogram of the 105,000 x g supernatant. Fraction A showed a sigmoidal response to the substrate concentration of guanine and its apparent Km value was 18pM. 3. Fraction B was the second-eluted peak from the column and was stained as a diffuse hand on the clcctropherogram which showed intermediate migration through the gel. Fraction B revealed a hyperbolic response to the substrate concentration of guanine and its apparent Km value was 22pM. 4. Fraction C was the lastly eluted fractions from the column and was separated into two diffuse bands on the gel which showed the most rapid migration through the gel. Fraction C revealed a hyperbolic response to the substrate concentration of guanine and its apparent Km value was 12pM. 5. All fractions were unaffected at O.lmM concentration of xanthine, hypoxanthine, NH., allantoin, GMP, GDP and GTP

Effect of ginseng on lipid metabolism(The Effect of Ginseng on Lipid Metabolism of Chicks)

The effect of long term administration of ginseng extract on lipid metabolism of chicks was studied. The chicks were fed for 24 months with the di t containing ginseng extract (0.4gm/kg body weight). From blood serum. the neutral fat was extracted and analyzed by thin-layer chromatography and gas chromatography. The results are as follow. 1. The total fat content of serum was significantly increased by long term administration of ginseng. 2. The concentration of cholesterol ester was not different from control group compared with ginsengtreated group. but the concentratoin of cholesterol was greately increased by ginseng treatment. 3. The free fatty acid concentration of serum 'did not show any difference between control group and ginseng-treated group but the concentration of triglyceride was significantly increased by long term administration of ginseng. 4. The fatty acid composition of serum did not show any difference between control group and ginsengtreated group, It suggest that the lipid metabolism of chicks might be greately influenced by long term administration of ginseng extract

On 5 nucleotidase and its isoenzymes in liver tissue of rat

51-Nucleotidase is an intrisic membrane glycoprotein which has been shown in a variety of cell types to be an ectoenzyme_ It is widely used as a plasma membrane marker in subcellular fractionation studies and used in monitoring cell membrane changes during neoplastic transformation. It has been reported as that 5ιnucleotidase has broad specificities for nucleoside 5' -monophosphate Attempts have been made for such a reason to resolve this enzyme into isoenzymes, but in most cases evidence for isoenzymes were not obtained. The present investigation was carried out to prepare lipid free 5ιnucleotidase from rat liver and to study its properties and isoenzymes with the following results. 1. Lipid-free 5ιnucleotidase obtained by Sephadex G-200 gel filtration or DEAE-cellulose chromatography from rat liver was resolved into two isoenzyme bands by polyacrylamide gel disk electrophoresis. 2. 5'-Nucleotidase was purified about 45 folds from rat liver homogenate with 42% yield by nbutanol extraction and gel filtration, but the enzyme isoenzymepurified by the treatment of n-butanol extraction and DFAE'callulose chromatography showed the 11 folds of purification with 5% yield. 3. The pH optimum and KM values of the two differently purified enzymes were identical; pH 7.5, O. 33mM, respectively. 4. The enzymes showed different pattern in the effect of EDTA; 5ιnucleotidase purified by gel filtration method revealed strongly inhibited activity, in contrast with that by DEAE-cellulose chromatography. 5. Effects of metal ions(Ca",Mg & Mn") on the activity of 5ιnucleotidase by two procedures used were similar to that by microsomal 5'-nucleotidase. 6. The effects of detergents (Triton X-IOO, sodium deoxycholate and sodium dodecylsulfate) on the activity of the enzymes were prominently different; the enzyme by gel filtration showed increased activity, while that by DEAE'cellulose chromatography, de' creased activity. The KM value , however was not altered, while Vmax, change

Electrophoretic study on the changes of serum protein fractions in Korean women -the effects of age, oral contraceptive, and intrauterine contraceptive device

Tbe serum levels of total protein, albumin, electrophoretic fractions from 610 Korean women, 21 to 50 years old, were measured to determine their rela~ tion to age, and the effects of oral contraceptive, Norinyl and intrauterine contraceptive deγice， Lippes Significant decrease in total protein and albumin concentrations were observed in oral contraceptive and IUD users, and rapid decrease in total protein was observed in oral contraceptive users than in IUD users. 껴 globulin was slightly increased in both groups, and r-blobulin was decreased in oral contracptive users. No significant change in ai-and a,-globulin concentratione was noted in both groups

Properties of 5'-Nucleotidase from Rat Liver Cell Plasma Membrane

5'-Nucleotidase bas been shown to be localized in the plasma membranes of various cell types. It is widely used as a marker enzyme for plasma membrane in subcellular fractionation. It has been reported as well that 5' -nucleotidase has broad specificities for nucleotide 5'-monophosphates. Attempts have been made for such a reason to resolve this enzyme into isoenzymes, but in most cases evidences for isoenzymes were not obtained. 5'-Nucleotidase has been reported recently to Increase in its activity upon aging the plasma membrane fraction in vitro at low temperature. A mechanism involved for the increase has been put foward in effect that a regulatory substance is bound to the enzyme, forming a latent 5f-nucleotidase. The present investigation was carried out to prepare membrane-free 5'- nucleotidase from rat liver plasma membrane and to study the kinetic properties of the enzyme. Attempts also were made to resolve 5'nucleotidase into isoenzymes and to confirm and characterize the proposed regulatory substance. The results obtained are summarized as follows: I. Membrane-free 5' -nucl eotidase obtained by sonification from rat liver plasma membrane was resolved into two isoenzyme bands by disk electrophoresis on the polyacrylamide gel. The slow-moving isoenzyme was major component whereas the fast-moving Isoenzyme, a minor component. 2. Membrane-free 5f -nuclcotidase compnzmg the major isoenzyme was purified about 12-fold from rat liver homogenate by differential centrifugation and gel filtration on Sephadex G-200 column equilibrated with 20mM sodium deoxycholate-20mM Tris, pH 9.2. 3. Activation of membrane·free 5f-nucleotidase was brought about to the same extent(5%) by the much lower concentration of MnH (0. 01-0. ImM) and by much higher concentration of MgH(IOmM). 4. The enzyme was strongly inhibited by EDTA and recovered completely by the addition of about 5 time as much MgH in molar concentration as EDTA. 5. The enzyme was strongly inhibited by ADP and to a much less extent by GDP and GTP. Purine nucleosides did not affect the enzyme activity. 6. The pH optimum and KM value of the enzyme were 8. a and O. 15mM respectively. 7. Phospholipid was detected in the membrane-free 5f-nucleotidase and the enzyme was activated by widely used detergents such as sodium dodecyl sulfate (SDS) , sodium deoxycholate(SDC) and Triton x-roo. Vma x of the enzyme was increased about 2-fold by addition of 5mg of SDC and 0.2 I' moles of MnH to 2ml of the enzyme assay system, but KM value of the enzyme was not altered. And the slope of the decrease in enzyme activity with time due to thermal denaturation at 30°C was greater in the SDC and Mnt t-containing assay system than in the control assay system. Based on the above results, it was discussed that the phospholipid bound to 5' -nucleotidase could be regulatory substance which could be released by the detergents, most effectively by SDC combined with MnH , and by incubation itself, causing the enzyme to increase in activity

Effects of EDTA on the Activities of Marker Enzymes for Membranes from Rat Liver.

For the examination of effects of EDTA on the activities of membrane marker enzymes, plasma membrane. mitochondria and microsomal membrane were partially purified from rat liver. O. 1M EDTA solntion buffered with 5mM Tris (pH 7.4) was added to these su bcellular particle suspensions to be 1mM or added directly to the enzyme assay system to survey the effects of EDTA on the enzyme activities under the varying conditions. The results obtained are as follows: 1. 5'-Nucleotidase and p-hydroxvbutyric dehydrogenase were enriched io.s-reu in the plasma membrane fraction and 8. Lfold in the mitochondrial fraction, respectively through differential centrifuga· tion followed by discontinous sucrose density gradient. The activities of (MgH+Na++K')-ATPase and glucose-fiephosphatase were slightly elevated by the presence of 1mM EDTA in the enzyme preparations. 2. According to kinetic study, the apparent Km for 5' -nucleotidase was 0.38mM and 5'-nucleotidase was inhibited competitively by EDTA. The enzyme activity was suppressed up to around 80% in the presence of O. 1M EDTA, but no further inhibition was observed beyond this concentration, and thus the rest of 20% is supposedly caused by other nonspecific phosphatases, 3. A modified method was attempted to establish determination of 5' -nucleotidase, taking advantages of selective inhibition of this enzyme by EDTA. The activity of phosphatases other than this enzyme is measured in the presence of O. 1M EDTA and the enzyme assay is once repeated but in the absence of EDTA, thus the difference of above two assayed activities would be the proper activity of 5' -nucleotidase, 4. Despite p-hydroxbutyric dehydrogenase (P-HBD) was actually inhibited by the presence of O.OlmM EDTA in the enzyme assay system, this mitochondrial enzyme activity, however, increased considerably when it was measured with the mitochondrial preparation with 1mM EDTA which was diluted to O. 01mM in the enzyme assay system. This apparent contradiction can be explained on the following assumption. The mitochondrial membrane structure is impaired by EDTA through the chelation of Cat" bound to membrance, rendering them vulnerable to the hypotonicity of enzyme assay medium. which results in the ruptured structures of mitochondrial membrane, offering much enhanced surface area with exposed enzyme. The resulting increased enzvme activity could well cover its inhibition. This presumption was proved by appropriate experimental design using intact mitochondria and fragmented one by sonication. 5. The ratio of intact mitochondria to ruptured Doe in mitochondrial preparation could be easily measured by the increase in p-HBD activity in the presence of 1mM EDTA, as compared with that of the sample without EDTA

Changes in the Activites of 5'-Nucleotidase relating to the Structural Change of the Membrane

Assay of 5'-nucleotidase is a biochemical tool to identify the plasma membrane in subcellular fractlonationc fy-Nucleotidase was reported to be activated by Mg" ion but the authors observed that the degree of activation depended on the structural state of the plasma membrane. This experiment was performed to study further in detail the change in 5'-nucleotidase activity relating to the chan· ge of the structural state of the plasma membrane. The results obtained are summarized as follows: 1. Rat livers were homogenized in O. 25M sucrose and the homogenate was fractionated into 2000 X g fraction, mitochondrial fraction and microsomal fraction according to the particle size of the plasma membrane. The activation effect of Mg" ions on 5'-nucleotidase was tested on each fraction. The 'effect was most pronounced in the microsomal frac- lion and the most effective concentration of Mg" 'ions was 10mM. 2. Microsomal vesicles increased in size in the presence of lOmM Mg" ions in the assay system of '5'-nucleotidase, which was confirmed by Tyndall effect and electron microscopy. 3. Microsomal vesicles were coagulated in the presence of 10mM Ca* ions in the assay system of '5'-nucleotidase, which was confirmed by Tyndall . effect and electron microscopy and Ca" ions inhibited 5'-nucleotidase about 30% at the concentration 'of 10mM. 4. From Tyndall effect of the microsomal vesicles it was shown that the vesicles were slowly enlarged in the hypotonic assay system at 30'C, but the vesicles did not increase in size at 16'C. 5. When the activities of 5'-nucleotidase were 'measured by starting the enzyme reaction with the .substrate, the activities of the enzyme were increased with increasing incubation period of the en' zyme preparation in the enzyme assay buffer at 30'C 'before addition of the substrate. From this result it is concluded that the activities of 5' -nucleotidase 'should be measured by starting the enzyme reaction with the enzyme preparation. 6. The activity of 5'-nucleotidase in the micro' somal fraction was increased with aging the mi- crosomal fraction in the refrigerator at 4 'C, which was accompanyed by the increase in lipid peroxide. Thus the increase in the enzyme activity of the microsomal fraction was about 40% after 4 days' aging. The activation effect of Mg" ions on 5'-nu. cleotidase was decreased with aging