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Purification, Characterization and Some Kinetic Properties of Fructose

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Purification, Characterization and Some Kinetic Properties of Fructose Yeditepe Medical Journal 2009;(11): 202-214 Purification, Characterization and Some Kinetic Properties of Fructose 1,6 Bisphosphate ABSTRACT Aldolase from Human Placenta Fructose–1,6–bisphosphate aldolase (E.C. 4.1.2.13) is a major glycolytic enzyme found in most cells. Fructose-1,6- bisphosphate aldolase reversibly catalyses Đnsan Plasentasından the cleavage of fructose 1,6-bisphosphate Fruktoz 1,6-Bisfosfat into the triose phosphates: D- glyceraldehyde phosphate and Aldolaz’ın Saflaştırılması, dihydroxyacetone phosphate. In this Tanımlanması ve Bazı study, we wanted to examine the Kinetik Özellikleri presence of aldolase in healthy human placenta and then topurify the enzyme. We also determined the optimum conditions (time, pH, and temperature) of enzyme assay measurements. With this Neşe Hayat AKSOY, PhD procedure, we determined the specific Department of Biochemistry, Faculty of Medicine, activity of placental aldolase as 831.90 Hacettepe University, Turkey mU/mg protein and aldolase was purified about 40.5 fold from healthy human placenta. It was demonstrated that the molecular weight of human placental Pakize DOĞAN, Prof. Dr. aldolase was 160 kD. Department of Biochemistry, Faculty of Medicine, Hacettepe University, Turkey In this study, substrate kinetics were also examined. Enzymatic assays were performed and substrate kinetic properties were detected and Vm value of healthy placental FBPA was determined as Corresponding Author 1769.513 ± 200.322, and Km as 20.003 ± 4.497 mM. Pakize Doğan, Prof. Dr. Keywords: Fructose 1,6 bisphosphate Department of Biochemistry, Faculty of Medicine, aldolase, Purification, Placenta, Kinetics Hacettepe University Ankara/Turkey E-mail : [email protected] 202 Yeditepe Medical Journal 2009;(11):202-214 Aksoy N.H. et al ÖZET from neuronal tissues, brain and smooth Fruktoz–1,6–bisfosfat aldolaz (E.C. muscle (1–6, 8, 10, 14). 4.1.2.13) pek çok hücrede bulunan major bir glikolitik enzimdir. Fruktoz–1,6– Aldolases are classifed as Class I and bisfosfat aldolaz geri dönüşümlü olarak Class II aldolases depending on their fruktoz–1,6–bisfosfat’ın iki trioz fosfata; requirement of divalent ions in catalysis. D-gliseraldehit 3-fosfat ve For their catalytic activity, Class I dihidroksiaseton fosfat’a bölünmesini Aldolases are not depend on cations and katalizler. found in higher animals and plants. Class I Aldolases form a “Schiff Base” with the Bu çalışmada amacımız sağlıklı insan substrate and are homotetramers (40.000 plasentasında aldolaz enziminin varlığını Da) (1-3). Class II aldolases are incelemek, ve enzimi saflaştırmaktır. Bu homodimers (80 kDa), found in bacteria çalışmaya ek olarak enzim aktivitesi tayini and fungi and require a metal cofactor için kullanılan yöntemin optimum şartlarını such as Zn+2 for catalysis (1–3, 7–14). (zaman, pH, ve sıcaklık) da tayin etmektir. Bu işlemde plasental aldolaz’ın spesifik Purification and definition the aktivitesi 831.90 mU/mg protein olarak characteristics and properties of human bulunmuş ve enzim sağlıklı insan placental FBPA have not been reported plasentasından 40.5 kere saflaştırılmıştır. previously. In our study, we wanted to Moleküler ağırlığı 160 kD olarak tespit examine the presence of aldolase in edilmiştir. Çalışmada sübstrat kinetiği de healthy human placenta and then to purify incelenmiştir. Enzimatik assay ve sübstrat the enzyme. We also determined the kinetiği özellikleri incelendiğinde Vm optimum conditions (time, pH, değerinin 1769.513 ± 200.322 mM ve Km temperature) of enzyme assay method. değerinin ise 20.003 ± 4.497 mM olduğu Consequently, advanced kinetic researchs bulunmuştur. had been optimised. MATERIALS AND METHODS Anahtar Kelimeler: Fruktoz 1,6 bisfosfat aldolaz, saflaştırma , plasenta, kinetikler Materials: All reagents used were of analytical grade and were obtained from INTRODUCTION Sigma Co. (St Louis, MO, USA). Fructose-1,6-bisphosphate aldolase (D Fru-1,6-bisphosphate D glyceraldehyde-3- Purification Procedures P-lyase; FBPA; E.C. 4.1.2.13) catalyses the reversible cleavage of fructose-1,6- Step 1. Preparation, extraction and bisphosphate (FBP) to dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3- homogenization of tissues phosphate (GAP) in the glycolytic pathway of prokaryotic and eukaryotic organisms. Human placental aldolases were purified FBPA is also an essential enzyme in the according to Penhoet method (15). All reversible gluconeogenesis and the purification procedures were carried out at fructose metabolic pathways (1–9). +4ºC. Three major tissue-specific types of The fresh placenta is obtained from aldolase isozymes are known in higher Hacettepe University, Gynaecology and organisms commonly referred toas: Type Obstetrics Department. Tissues were A, excreted from the classical muscle and washed, and perfused with cold buffer red blood cells enzyme, Type B, from containing; 50mM Tris-HCl, 5 mM EDTA liver, kidney and small intestine, Type C, and 4 mM mercaptoethanol and isotonic 203 Yeditepe Medical Journal 2009;(11):202-214 Aksoy N.H. et al NaCl (0,95% ml), pH=7.5 at 4ºC. They Step 4. Substrate elution from were weighed, minced into small pieces phosphocellulose column and suspended in 3 volumes (v/w) of the buffer at +4ºC. Then the sample was The desalted fraction was applied to a homogenized for about 10 strokes by phosphocellulose column for purification of using a Teflon homogenizer in an ice bath aldolase. The fractions were absorbed at and centrifuged for 1 hour at 14.000 × g, 0.5 ml/min onto a column of and +4ºC. The precipitate was discarded phosphocellulose pre-equilibrated with and the clear supernatant was removed buffer. The column was washed with the for ammonium sulphate fractionation. buffer until the A280 decreased to baseline, (narrow red band visible on the Step 2. Ammonium sulphate [(NH4) 2 column and OD of the efluent at A280 nm drop below 0.1) and then the substrate SO 4] fractionation elution was carried out with stock buffer In order to obtain 45% ammonium containing 2.5 mM Fru–1,6-P2. After this sulphate [(NH4) 2SO 4] concentration, process, enzyme was eluted with 50 mM 26.20 g of solid [(NH4) 2SO 4] was added to Tris-HCl, 5 mM EDTA pH 7.5 buffer, 100 ml of supernatant and waited for 1 containing 1 mM substrate. hour period then centrifuged at 14 000xg for 1 hr, at +4ºC, supernatant was Protein and enzyme activity obtained and precipitate was discarded. At determinations were performed at all the second step of [(NH4) 2SO 4] along the line of gel filtration and fractionation (65%); for each 100 ml phosphocellulose column chromatography supernatant fluid, 12.5 g [(NH4) 2SO 4] stages. was added and the procedure was repeated. For, this time, supernatant was Polyacrylamide Gel Electrophoresis discarded, the precipitate was collected, and dissolved in 10 mM Tris-HCl-EDTA SDS-PAGE with polyacrylamide gel was buffer containing 1 mM EDTA, pH= 7.5 performed according to the modified and any insoluble material is discarded by Laemmli method (17). Purified enzyme centrifugation. was applied to SDS-PAGE electrophoresis inorder todetermine the molecular weight Protein (18) and activity (16) and purity. Samples were run on a 7.5 % determinations of the precipitates and (w/v) acrylamide separating gel and a 4.5 supernatants were performed at all along % stacking gel. Proteins on gels were the line of extraction and fractionation. stained with Coomassie Brilliant blue R– 250. Chromotographic Procedures Enzyme Assay Step 3. Sephadex G–25 gel filtration The quantitative estimation of aldolase for desaltation activity present in the extracts are determined by measuring the rate of For the desaltation, the dissolved cleavage of fructose 1,6 bisphosphate precipitate solution obtained after 65% spectrophotometrically. A unit of aldolase ammonium sulfate [(NH4) 2SO 4] activity was defined as that amount of application was passed through a column enzyme which catalyses the cleavage of 1 of Sephadex G-25. The column was µmole of substrate per minute at 37ºC equilibrated with running buffer. The under conditions of assay. The specific sample volume was 20 ml and flow rate activity was defined as the number of range was 2 ml/min. At the end of the activity units per milligram of protein. The application all samples have been desalted ubstrate cleavage rate was determined (Data not shown). spectrophotometrically by measuring 204 Yeditepe Medical Journal 2009;(11):202-214 Aksoy N.H. et al hydrazone formation of product with Specific activity of placental aldolase was dinitrophenyl hydrazine (DNPH) (16). determined as 831.90 mU/mg protein. Summary of the purification steps are presented at Table 1. Protein Determination During the purification of FBPA procedure, Pure enzyme samples were applied to protein determination was performed at SDS-PAGE electrophoresis for control of 280 nm, corrected for nucleic acid purity and in order to determine the impurities according to Warburg and molecular weight (17). We demonstrated Christian (19). The A280 method takes that the purity of enzyme samples and advantage of the absorbance of light at also verified molecular weight of human 280 nm by the aminoacids Tyr and Trp. placental aldolase was 160 kD (Figure 2). Amount of the protein in the samples was Samples were run on a 7.5% (w/v) determinated with Lowry Method that acrylamide separating gel and a 4.5% slightly modificated (18). The reactions stacking gel. The subunit molecular mass result in a strong blue color, read at 750 was determined as 40 kDa. Therefore it nm. Determination was run triplicate for was observed that the human placental all samples. aldolase was a homotetramer (Figure 3). Kinetic Studies pH optimization All experimental conditions were adjusted The effect of pH on the aldolase activity at 37ºC, and pH= 7.4 which were was determined by using 50 mM FBP with optimum enzyme assay conditions (16). the following buffers (50 mM) at the indicated pH; acetate buffer (pH 5.0 and Non-linear regression analysis module of 5.5), phosphate buffer (pH 6.0 and 6.5), Systat 11 statistic program was used to Tris–HCl buffer (pH 7.0–8.5).
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