13th balkan biochemical biophysical days & meeting on metabolic disorders’ programme & abstracts
STEADY-STATE KINETICS OF RAT INTESTINAL BUTYRYLCHOLINESTERASE
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- THE EFFECT OF FLUDARABINE ON MYELOPEROXIDASE ACTIVITY
STEADY-STATE KINETICS OF RAT INTESTINAL BUTYRYLCHOLINESTERASEYıldız Ö., Çokuğraş A.N., Özer N.Department of Biochemistry, Faculty of Medicine, Hacettepe University Ankara / TurkeySteady-state kinetics of soluble isoform of butyrylcholinesterase (BChE; E.C.3.1.1.8.), purified from rat small intestine, were determined when acetylthiocholine (ATCh), propionylthiocholine (PTCh) butyrylthiocholine (BTCh) and benzoylcholine (BzCh) were used as substrates. The plots of velocity versus substrate concentrations did not give the normal hyperbolic Michaelis-Menten curves for all substrates studied. [S]0.75 / [S]0.50 ratios were found to be approximately 4 for ATCh, PTCh, BTCh, which indicate substrate activation, whereas to be 2 for BzCh, which indicates subtrate inhibition at high substrate concentrations. So, steady-state data were fit to the equation for excess substrate activation / inhibition.The calculated parameter b in this equation reflects the efficiency of product formation from ternary complex (SES). When b>1, there is substrate activation. Rat BChE purified from soluble fraction of the intestine showed marked substrate activation with acyl-choline substrates, ATCh, PTCh and BTCh as reflected in b values obtaining as 2.75, 2.15 and 1.63, respectively. But, for BzCh, we found b value as 0.426. If b is less than 1, there is substrate inhibition. As a measure of catalytic efficiency, kcat / Km values were determined as 16 210, 25 650, 46 150 for ATCh, PTCh, BTCh, respectively. When the catalytic efficiencies were compared, soluble isoform of rat intestinal BChE became increasingly efficient as the size of acyl portion of the substrate increases; BTCh >PTCh >ATCh. Differently, the enzyme showed subtrate inhibition for BzCh and kcat / Km values was found to be 21 190. It can be said that BChE is equally efficient with BzCh and PTCh. The inhibitory effect of Triton X-100 on rat intestinal and human serum BChEs were compared. It is found that Triton X-100 at higher concentrations than its critical micellar concentration (70mM) inhibited 30% of the rat intestinal BChE activity whereas 15% of human serum BChE activity when BTCh was used as substrate. P264 ANTIOXIDANT CAPACITIES OF BARK EXTRACTS FROM Aesculus hippocastanum L. Erkin AYDIN 1, Nursen ÇORUH 2 1 Graduate School of Natural and Applied Sciences, Biochemistry Division, Middle East Technical University, 06531, Ankara, TURKEY 2 Department of Chemistry, Middle East Technical University, 06531, Ankara, TURKEY e-mail: ncoruh@metu.edu.tr Aesculus hippocastanum L., commonly known as horse chesnut tree is an ornamental and medicinal tree widely found in the flora of Turkey. This plant has a long history of use in different medicinal preperations. Saponins, flovanoids and coumarines are the most significant examples. The aim of this work was preparation of several extracts from the barks of horse chestnut trees, then to decide on their inhibitory effects over microsomal lipid peroxidation. After the antioxidant capacity of crude extracts were determined, the effective components of the extracts were isolated through simple separation techniques. The ground bark was extracted with a rotary evaporator, using methanol, ethyl acetate or water as solvents in 1:6 bark to solvent ratio. Each extract was tested for their antioxidant capacities through the inhibition of lipid peroxidation. Thiobarbutiric acid (TBA) test was applied for the measurement of lipid peroxidation. Lipid peroxidation was induced by Fe(II) on the microsomes isolated from sheep liver. 50 percent inhibitory concentrations (IC50) of the extracts were found as 0.008 mg/ml for ethyl acetate extract, and 0.022 mg/ml and 0.012 mg/ml for the water and methanol extracts. Ethyl acetate extract was observed to be the most efficient antioxidant among the others, a simple chromatography method was applied to separate its individual antioxidant components. Lipophilic sephadex (LH-20) was used as the column material and column was eluted with methanol. Antioxidant capacity of the three seperable individual isolates were determined as IC50 values of 0.080 mg/ml, 0.140 mg/ml, and 0.050 mg/ml according to the order of elution. The isolate with the most potent antioxidant capacity was identified as esculetin (6, 7-dihydroxy coumarin) using spectroscopic (UV-VIS, IR, NMR) techniques. Ethyl acetate extract was deduced as having higher antioxidant capacity. A simple LH-20 chromatography revealed an important antioxidant component from the bark of Aesculus hippocastanum L. which was identified as esculetin. P265 THE EFFECT OF FLUDARABINE ON MYELOPEROXIDASE ACTIVITYA. Lale DOĞAN1, Ayça DOĞAN2, Övünç DÜZGÜNÇINAR2, Ediz DEMİRPENÇE2 1Hacettepe University Institute of Oncology, Department of Basic Oncology, and 2Hacettepe University Faculty of Medicine, Department of Biochemistry, Ankara. Fludarabine is an adenine nucleoside analog that induces leukemic cell apoptosis and shows high efficacy in the treatment of chronic lymphocytic leukemia. However, severe bone marrow supression, notably anaemia, thrombocytopenia and neutropenia, has been reported in patients treated with Fludarabine. An impairment of the neutrophil function in addition to neutropenia would certainly worsen the situation. Neutrophils are phagocytic cells that contain myeloperoxidase. During phagocytosis, myeloperoxidase catalyzes the formation of hypochlorous acid from hydrogen peroxide and chloride ion. This is the main microbicid system in phagocytes. The aim of our study was to evaluate the effect of Fludarabine on neutrophil function through myeloperoxidase activity. Cultured peripheral blood leukocytes and HL-60 cells were used in this study. Leukocytes were isolated, put into culture and incubated with or without Fludarabine for 48 hours. At the end of the incubation period, myeloperoxidase activities were measured. To assess a possible dose-dependent direct effect of Fludarabine on myeloperoxidase activity, HL-60 cells were used. Myeloperoxidase activity was measured by a spectrophotometric method using tetramethyl benzidine as synthetic substrate. Enzyme activity was expressed as unit/mg protein. Experiments were performed in triplicate and results are given as mean±SD. Myeloperoxidase activity was significantly increased in leukocytes incubated with Fludarabine (control 1,58±0,18, Fludarabine 7,80±0,85). This could be due to a direct effect of Fludarabine on the enzyme or an increase in myeloperoxidase expression. Since HL-60 cells contain substantial amount of myeloperoxidase, we used them to evaluate the dose-dependent direct effect of Fludarabine on the enzyme activity. Fludarabine was added at concentrations ranging between 10 M and 2 mM. There was no significant change in myeloperoxidase activity at any Fludarabine concentration. It has been shown that hypochlorous acid (product of the reaction catalyzed by myeloperoxidase) triggered apoptosis. We suggest that increased myeloperoxidase expression might be involved in the mechanism of Fludarabine-induced apoptosis. P266 EFFECTS OF TWO SYNTHETIC COMPOUNDS AS ANTIDOTES FOR CHLORSULFURON IN CORN Directory: 2003 2003 -> Tr-41. 4-03-05-024 Telecommunications 2003 -> Review of some tsunamis in canada 2003 -> The skill of multi-model seasonal forecasts of the wintertime North Atlantic Oscillation 2003 -> 2930 Kerry Forest Parkway 2003 -> Canadian Interuniversity Sport (cis) Athletes of the Week 2003 -> Senior Airman Jason Plite Hometown 2003 -> Technical announcements: My apologies for the comparison pages; they were 2003 -> Pool Play Round 1 9: 20: 00 am 2003 -> 2004 Division II men's Golf Championships Final Round Results deland, fla 2003 -> Go Figure Edited by Kostya Kennedy and Mark Bechtel 645 Passing yards, an ncaa division II record, by University of Indianapolis quarterback Matt Kohn, in a 59-52 victory over Michigan Tech Download 1.91 Mb. Share with your friends:
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