Purification and Kinetic Properties of 6-Phosphogluconate ...

The Protein Journal, Vol. 24, No. 5, July 2005 ( 2005) DOI: 10.1007/s10930-005-6750-z

Purification and Kinetic Properties of 6-Phosphogluconate Dehydrogenase from Rat Small Intestine Deniz Ceyhan,1 Ali Danis¸ an,1 I. Hamdi O¨g˘u¨s¸ ,1 and Nazmi O¨zer1,2

6-Phosphogluconate dehydrogenase (6PG) was purified from rat small intestine with 36% yield and a specific activity of 15 U/mg. On SDS/PAGE, one band with a mass of 52 kDa was found. On native PAGE three protein and two activity bands were observed. The pH optimum was 7.35. Using Arrhenius plots, Ea, DH, Q10 and Tm for 6PGD were found to be 7.52 kcal/ mol, 6.90 kcal/mol, 1.49 and 49.4C, respectively. The enzyme obeyed ‘‘Rapid Equilibrium Random Bi Bi’’ kinetic model with Km values of 595 ± 213 lM for 6PG and 53.03±1.99 lM for NADP. 1/Vm versus 1/6PG and 1/NADP plots gave a Vm value of 8.91±1.92 U/mg protein. NADPH is the competitive inhibitor with a Ki of 31.91±1.31 lM. The relatively small Ki for the 6PGD:NADPH complex indicates the importance of NADPH in the regulation of the pentose phosphate pathway through G6PD and 6PGD. KEY WORDS: Ea; glucose-6-phosphate dehydrogenase; DH; Ki values; NADPH inhibition; pH optimum; purification; Q10; rat small intestine.

against oxidative agents by transferring its reductive power to glutathione disulfide (GSSG) via glutathione disulfide reductase (Eggleston et al., 1974; Levy, 1979; Rosemeyer, 1987). Several reports are available regarding the purification of 6PGD from tissues of many species by gel filtration, ion exchange and/or affinity chromatography and preparative gel electrophoresis (Procsal and Holten, 1972; Dyson et al., 1973;

1. INTRODUCTION 6-Phosphogluconate dehydrogenase (6-GPD, EC 1.1.1.44, 6-Phosphogluconate; NADP oxidoreductase) is widely distributed among microorganisms, plants and in different tissues of animals (Villet and Dalziel, 1969; Pearse et al., 1973; Toews et al., 1976; Weisz et al., 1985; Krepinsky et al., 2001; Beydemir et al., 2004). It catalyzes the second step of the pentose phosphate pathway (PPP). PPP produces NADPH, pentose phosphates necessary for nucleotide biosynthesis and serves as the route of entry of 3–5 carbon sugars to the glycolytic pathway. NADPH donates protons and electrons for a variety of reductive reactions, including fatty acid and cholesterol biosynthesis. NADPH also has very important functions in the protection of the cell 1

2

Abbreviations: 6PG, 6-Phosphogluconate; 6PGD, 6-Phosphogluconate dehydrogenase; PPP, Pentose phosphate pathway; GSH, Glutathione, reduced; GSSG, Glutathione disulfide, oxidized glutathione; GSSGR, Glutathione disulfide reductase; GSTs, Glutathione S-transferases; NADPH, Nicotinamide adenine dinucleotide phosphate, reduced form; NADP+, Nicotinamide adenine dinucleotide phosphate, oxidized form; NADH, Nicotinamide adenine dinucleotide, reduced form; NAD+, Nicotinamide adenine dinucleotide, oxidized form; e-ACA, e-Aminocaproic acid; EDTA, Ethylenediamine tetraacetic acid; PAGE, Polyacrylamide gel electrophoresis; SDS/PAGE, Sodium dodecyl sulfate/polyacrylamide gel electrophoresis; 2-ME, 2-Mercaptoethanol; 2¢,5¢-ADP-Sepharose 4B, 2¢,5¢-Adenosine diphosphate-Sepharose 4B; DEAESepharose FF, Diethylaminoethyl-Sepharose Fast Flow.

Department of Biochemistrry, Faculty of Medicine, Hacettepe University, 06100, Ankara, Turkey. To whom correspondence should be addressed: Nazmi O¨zer Tel.: +90 312 305 2162; Fax: 790 312 311 0588; E-mail: naozer@ hacettepe.edu.tr

293 1572-3887/05/0700-0293/0 2005 Springer Science+Business Media, Inc.

Ceyhan, Danis¸ an, O¨g˘u¨s¸ , and O¨zer

294 Betts and Mayer, 1975; Bridges et al., 1975; Cottreau and Boivin, 1975; Toews et al., 1976; Weisz et al., 1985; Rosemeyer, 1987; Krepinsky et al., 2001; Beydemir et al., 2004). The eukaryotic 6PGD usually consists of two subunits each having a molecular mass of about 40–52 kDa (Procsal and Holten, 1972; Silverberg and Dalziel, 1973; Pearse and Rosemeyer, 1975). The subunits of 6PGD show structural and functional asymmetry and each subunit of the dimer has three domains: a ba-b domain binds NADP; an all a domain provides much of the dimer interface; the C-terminal tail burrows into the second subunit (Adams et al., 1991; Rippa et al., 2000). In bacteria and in insects higher and lower subunit molecular masses and dimeric and/or tetrameric forms have been reported (Tsai and Chen, 1975; Williamson et al., 1980; Stournaras et al., 1983; Yoon et al., 1989; Bianchi et al., 2001). It is clear that orally introduced drugs, food preservers, etc. first meet the detoxifying enzymes found in the digestive tract, such as glutathione-Stransferases (GSTs) and modified by conjugation with acceptor molecules (Chasseaud, 1979; Tahir et al., 1988; O¨zer et al., 1990). GSTs require the tripeptide glutathione (GSH) (Chasseaud, 1979). The tripeptide, GSH and its disulfide, GSSG, comprise the major, low-molecular-weight thiol/ disulfide redox buffer of most cells (Meister et al., 1981) and the level of GSH is strictly controlled by glutathione disulfide reductase (GSSGGR) at the expense of NADPH which is a product of glucose-6-phosphate dehydrogenase (G6PD) and 6PGD activities. In toxicity investigations, the rat is the most commonly used animal. A limited number of reports for the intestinal detoxification enzymes; glutathione-S-transferases (GSTs), butyrylcholinesterase (BChE) and glucose-6-phosphate dehydrogenase (G6PD) exist but no report related to the rat small intestinal 6PGD purification exists in the literature (Tahir et al., 1988; O¨zer et al., 1990; Danisan et al., 2004; Yildiz et al., 2004). Studying the special features of 6PGD in detail will provide information directly about biosynthesis of some biomolecules and, indirectly about detoxification. The purification procedure for 6PGD from rat small intestine given below, involved Sephadex G-25 gel filtration, ion-exchange and affinity chromatographies and produced an enzyme of at least 95% purity with a specific activity of 15 U/mg protein and a yield of 36%.

2. MATERIALS AND METHODS 2.1. Chemicals Sephadex G-25, DEAE-Sepharose Fast Flow and 2¢,5¢-ADP Sepharose 4B were from PharmaciaLKB, Uppsala, Sweden; glycerol from Darmstadt, Merck, Germany; 2-mercaptoethanol (2-ME), 6-phosphogluconate (6-PG, cyclohexylammonium salt), b-nicotinamide adenine dinucleotide phosphate, (b-NADP+ and b-NADPH, oxidized and reduced forms, sodium salts), b-nicotinamide adenine dinucleotide (b-NAD+, lithium salt), e-aminocaproic acid (e-ACA), ethylenediamine tetraacetic acid (EDTA) from Sigma, St. Louis, USA. All other chemicals were standard products of Sigma or Aldrich, USA. 2.2. Purification of Rat Small Intestinal 6PGD In this study, small intestines were obtained from female rats killed for students’ laboratory coursework at Hacettepe University Medical School. All steps were performed at 4C. The small intestines were washed with ice-cold 5 mM phosphate buffer, pH 8.0, containing 5 mM 2-ME, 4 mM EDTA, 1 mM e-ACA and 1 mM MgCl2 (Buffer A). The small intestines were washed with Buffer A, weighed and chopped into 3–4 cm pieces and homogenized in three volumes of Buffer A (g/ ml) by using an Omni 5000 homogenizer at 30,000 rpm for 5 30 s with 1 min interval, in ice bath. The homogenate was centrifuged at 34,800 g for 45 min using Sorvall RC-5B centrifuge. The supernatant was recentrifuged at 105,000 g for 1 h using Beckmann L70 ultracentrifuge and the supernatant (12 ml; 12.42 Units) was applied onto a column (2.8 cm 20.0 cm) of Sephadex G-25 column preequilibrated with 5 mM phosphate buffer, pH 8.0, containing 5 mM 2-ME, 1 mM MgCl2 and 1 mM e-ACA (Buffer B). During sample application and elution, flow rates and fraction volumes were 48 ml/hr and 4 ml, respectively. The active fractions (34 ml, 9.23 Units) were combined and applied onto a column of DEAE-Fast Flow ion exchanger (2.4 cm 8 cm) preequilibrated with Buffer B. Following application of the sample, the ion exchange column was washed with Buffer B until the absorbance of the effluent at 280 nm was £ 0.01. 6PGD was eluted using 250 ml linear gradient of 5/150 mM phosphate buffer, pH 8.0 (The other components of the Buffer B kept constant). During

6-Phosphogluconate Dehydrogenase from Rat Small Intestine sample application, washing, and elution of the enzyme the flow rates and fraction volumes were 24 ml per hour and 3.5 ml, respectively. The fractions having 6PGD activity were collected (15 ml; 7.5 Units) and was loaded onto a 2¢,5¢-ADP Sepharose 4B column (1.6 cm 2.0 cm) preequilibrated and washed with Buffer B, until the absorbance of the effluent at 280 nm was £ 0.01. 6PGD elution was carried out with a 40 ml linear gradient of 0/250 mM (NH)2SO4 in Buffer B. During sample application, washing, and elution the flow rates and fraction volumes were 7.2 ml per hour and 2.6 ml, respectively. Glycerol (final 20%, v/v) was added to the eluted enzyme (5.2 ml; 4.45 Units), which was then distributed, as 1 ml aliquots, into Eppendorf tubes and stored at )20C.

295

the activity measurement medium, except enzyme, were brought to the desired temperature (20–58 C) and the reaction was initiated by the addition of the enzyme. The logarithm of the activities obtained were plotted versus 1/T (K) and the activation energy, activation enthalpy, Q10 and optimum temperature were calculated from the graph (Segel, 1975; Wilson, 1971). 2.6. Protein Determination Protein concentration in chromatographic fractions was estimated by measuring the absorbances at 280 nm. In pooled samples protein content was determined by the micromethod of Bradford, using bovine serum albumin as standard (Bradford, 1976).

2.3. Activity Measurements 6PGD activity was determined at 37C by the method of Beutler (Beutler, 1971). The conversion of NADP to NADPH was followed by monitoring the change in absorbance at 340 nm, using a Shimadzu 1601 spectrophotemeter (e 340= 6.22 m M)1cm)1). The reaction mixture (for routine activity determinations) contained 100 mM phosphate buffer, pH 7.7, 10 mM MgCl2, 0.1 mM NADP and a suitable amount of the enzyme. The assay was initiated by the addition of 6-PG to give a final concentration of 1.0 mM. One unit of enzyme activity was defined as the amount of the enzyme which converts one micromole of NADP to NADPH per min under defined conditions. 2.4. pH Optimum Determination To avoid buffer effects on the observed pH optimum phosphate buffers at different pH values and concentrations were prepared (Rosemeyer, 1987; Aksoy et al., 2001). At each pH activity was plotted vs [buffer]. The 6PGD activity at zero buffer concentration was determined by extrapolation. The extrapolated activities were used in constructing the pH-activity profile. 2.5. Determination of Ea, DH , Q10 and Optimum Temperature for 6PGD For the determination of activation energy, activation enthalpy and Q10, all the components of

2.7. Polyacrylamide Gel Electrophoresis (PAGE) Discontinuous PAGE (4% stacking gel and 7.5% separator gel: 0.75 70 100 mm) was carried out under nondenaturing conditions, essentially as described by Davies (Davies, 1964). Following electrophoresis, the gels were stained for either activity (Voupio et al., 1973) or protein (Davies, 1964). SDS/PAGE (4% stacking gel and 12% separator gel: 0.75 70 100 mm) was carried out and stained using Coomassie Brillant Blue R250 exactly as described by Laemmli (Laemmli, 1970). Both types of electrophoretic separation were carried out using the Mini protean II system of Bio-Rad (USA) at room temperature at 125 volts for 30 min.

2.8. Substrate Kinetics Substrate kinetics were determined at 37C in one-ml reaction mixture containing 100 mM phosphate buffer, pH 7.7, 10 mM MgCl2, and various concentrations of NADP and 6-PG. A matrix of substrate and coenzyme combinations between 10–160 lM (NADP) and 20–1280 lM (6-PG) was formed and the reactions were initiated by the addition of the enzyme. The activities were used in constructing Lineweaver-Burk and additional diagnostic plots to obtain Km and Vm values for substrate, coenzyme and Ki value for the inhibitor, NADPH (Cleland, 1979).


296 2.9. The Analysis of the Kinetic Data The kinetic data were analyzed and kinetic constants were calculated by means of the non-linear curve-fitting programme of the Statistica package (1999 Edition; Statsoft, Inc., USA).

3. RESULTS AND DISCUSSION 3.1. Purification of 6PGD from Rat Small Intestine 6PGD was purified from rat small intestine in successive steps involving fractional centrifugation, Sephadex G-25, ion-exchange on DEAE-Sepharose Fast Flow and 2¢,5¢-ADP Sepharose 4B affinity column chromatographies. At all steps the medium was supplemented with 2-ME as antioxidant (Bridges et al., 1975; Cottreau and Boivin, 1975; Rosemeyer, 1987; Scott and Abramsky, 1973; Silverberg and Dalziel, 1973; Weisz et al., 1985; Yoon, et al., 1989) EDTA and e-ACA were added for protection against proteases (Procsal and Holten, 1972; Rosemeyer, 1987; Yoon et al., 1989); glycerol or/and (NH4)2SO4 were added to preserve the enzyme in its active form (Bridges et al., 1975; Cottreau and Boivin, 1975; Pearse and Rosemeyer, 1975; Procsal and Holten, 1972; Rosemeyer, 1987; Villet and Dalziel, 1969; Weisz et al., 1985). Particulate matter in the initial homogenate was removed by centrifugation and the supernatant was applied to Sephadex G-25 column and at this step, 60

percent of protein was removed with a loss of 26 percent in activity (not shown). Chromatography on DEAE-Sepharose Fast Flow gave 13-fold purification with a loss of 19 percent in activity (Fig. 1). The overall yield at this step was 60 percent. Sephadex G-25, DEAE-Sepharose and affinity chromatographies (Fig. 2) resulted in 473 fold purification, 36 percent yield and the purified enzyme had a specific activity of 15 units/mg protein. A summary of the purification is presented in Table 1. The purified rat small intestinal 6PGD was stable for several months in concentrated solution and in the presence of glycerol or/and ammonium sulfate (Dalziel, 1969; Procsal and Holten, 1972; Cottreau and Boivin, 1975; Pearse and Rosemeyer, 1975; Villet and Weisz et al., 1985; Rosemeyer, 1987). The specific activity of 6PGDs obtained from the same and/or different sources shows great variability. The specific activities of 6PGDs obtained from mammalian sources (0.41 – 22.6 units/mg protein) (Betts and Mayer, 1975; Beydemir et al., 2004; Cottreau and Boivin, 1975; Dyson et al., 1973; Pearse and Rosemeyer, 1975; Procsal and Holten, 1972; Silverberg and Dalziel, 1973; Toews et al., 1976; Villet and Dalziel, 1969; Weisz et al., 1985) are lower than the specific activities of 6PGDs obtained from bacterial sources (20 – 60 units/mg protein) (Bianchi et al., 2001; Bridges et al., 1975; Krepinsky, et al., 2001; Pearse and Harris, 1973; Stournaras et al., 1983; Tsai and Chen, 1975; Yoon et al., 1989). It was shown that some bacterial 6PGDs prefer to use NAD as coenzyme; e.g. 6PGD

0.8

0.12

0.6

0.08

0.4

0.04

0.2

Protein, A280

Activity, ∆ A340/min; KP, M

0.16

0

0 0

25

50

75

100

125

Tube Number Fig. 1. DEAE-Sepharose Column Chromatography of Rat Small Intestinal 6PGD. Column dimensions 2.5 8 cm. Flow rate during application, wash, and elution was 24 ml/h. Fraction volume 3.5 ml. Absorption at 280 nm (light line); Activity (heavy line). Dotted line shows 250 ml linear gradient of 5/150 mM potassium phosphate.

0.2

Activity, ∆A340/min

0.25 0.1

0.20 0.15

0.1

0.10 0.0 0.05 0.0

0.00 0

10

20

30

Protein,A280 nm; (NH4)2SO4, M

6-Phosphogluconate Dehydrogenase from Rat Small Intestine

40

Tube Number Fig. 2. Affinity Chromatography of Rat small intestinal 6PGD on 2¢, 5¢-ADP-Sepharose. Column dimensions, 1.6 2 cm. Flow rate during application, wash, and elution was 7.2 ml/h; fraction volume of 2.6 ml. Absorption at 280 nm (light line); Activity (heavy line). Dotted line shows 40 ml 0/250 mM ammonium in 5 mM potassium phosphate buffer, pH 7.7.

obtained from Pseudomonas fluorescens have specific activities of 23 and 121 units/mg protein with NADP and NAD coenzymes, respectively (Stournaras et al., 1983). The specific activity of the rat small intestinal 6PGD, 15 units/mg protein, is comparable with the specific activities of 6PGDs obtained from other mammalian sources (Table 1). The great differences observed in the specific activities of 6PGDs may be the result of the origin of the enzyme (Betts and Mayer, 1975; Beydemir et al., 2004; Cottreau and Boivin, 1975; Dyson et al., 1973; Pearse and Rosemeyer, 1975; Procsal and Holten, 1972; Silverberg and Dalziel, 1973; Toews et al., 1976; Villet and Dalziel, 1969; Weisz et al., 1985) or may simply be explained by the variability of the inactive enzyme content in the enzyme preparations due to proteolytic degradation; e.g. for 6PGDs purified from sheep liver specific activities ranging from 6.6 to 18.7 units/mg protein were reported (Dyson et al., 1973; Silverberg and Dalziel, 1973; Villet and Dalziel, 1969). This situation was also observed in glucose-6-phosphate dehydrogenase preparations from

297

the rat small intestine (Danisan et al., 2004) and from the other sources such as human placenta, dog liver and human erythrocytes (Aksoy et al., 2001; Holten, 1972; O¨zer et al., 2001, 2002; Yoshida, 1966). The specific activity reported here for 6PGD might be lower than expected due to the high proteolytic activity in the rat small intestine (Table 1; Fig. 3a–c). Indeed, in native electrophoresis two distinct bands (one probably corresponding to dimeric and the other to monomeric form of the enzyme) and a diffuse band with no activity were observed on the native gel stained for protein (Fig. 3a, b). The inactive diffuse band seems as denatured enzyme because on the SDS / PAGE only one band with mass of 52 kDa was observed (Fig. 3c). It was shown that the specific activity of 6PGD depends on several factors such as the type of buffer, pH, [salt], NADP, RSH, mono- and divalent [cations] (Bridges et al., 1975; Cottreau and Boivin, 1975; Dyson et al., 1973; Pearse and Rosemeyer, 1974; Rosemeyer, 1987; Scott and Abramsky, 1973; Toews et al., 1976; Weisz et al., 1985). The active form of 6PGDs is either dimer or tetramer, depending on the source of the enzyme. All mammalian 6PGDs, reported so far, are dimeric and their subunit mass ranges from 38 to 65 kDa (Betts and Mayer, 1975; Beydemir et al., 2004; Pearse and Rosemeyer, 1975; Procsal and Holten, 1972; Silverberg and Dalziel, 1973; Toews et al., 1976; Villet and Dalziel, 1969; Weisz et al., 1985). On the other hand, the structures of 6PGDs isolated from other sources such as bacteria, fungi, or plants are either homodimeric or homotetrameric; subunit mass ranging from 31 to 70 kDa (Bianchi et al., 2001; Bridges et al., 1975; Krepinsky et al., 2001; Pearse and Harris, 1973; Scott and Abramsky, 1973; Stournaras et al., 1983; Tsai and Chen, 1975; Williamson et al., 1980; Yoon et al., 1989). The tetrameric structure is usually associated with lower subunit Mr; i.e. for Pseudomonas fluorescens and Schizosaccaromyces pompe 6PGDs have tetrameric structures with subunit masses of 31 and 38 kDa, respectively (Stournaras

Table 1. Purification of Rat Small Intestinal 6-Phosphogluconate Dehydrogenase

Purification step 105,000 g supernatant Sephadex G 25 eluate DEAE – eluate Affinity eluate

Volume (ml)

Activity (U/ml)

Protein (mg/ml)

T. Activity (Units)

T. Protein (mg)

12.0 34.0 15.0 5.2

1.040 0.270 0.500 0.855

31.960 4.560 0.640 0.057

12.42 9.23 7.50 4.45

383.47 155.00 9.60 0.294

Sp. Activity (U/mg Prot.) 0.032 0.060 0.781 15.120

Yield (%)

Purification fold

100 74 60 36

1.00 1.86 24.41 473.00

298


Fig. 3. Native (Spacer gels 4%; Separator gels 7.5%) and SDS/PAGE of Rat small intestinal 6PGD (Spacer gels 4%; Separator gels 12%). (a) Protein staining with coomassie brillant blue R-250 (native gel): Lane 1, Sephadex G-25 eluate, 5.3 lg; Lane 2, 105,000 g supernatant (4.9 lg); Lane 3, negative control; Lane 4 and 5, 2¢,5¢-ADP Sepharose 4B eluate (6.5 lg). (b) Activity staining: Lane 1, Sephadex G-25 eluate, 5.3 lg; Lane 2, 105,000 g supernatant (4.9 lg); Lane 3, negative control; Lane 4, 2¢,5¢-ADP Sepharose 4B eluate (6.5 lg). (c) Protein staining with coomassie brillant blue R-250 (SDS/PAGE): Lane 1 and 7, from top to bottom pointing at the positions of the standard proteins (Protein mixture total 10 lg): HSA, Human serum albumin (65 kDa); Ova, Eggalbumin (45 kDa); Try, Trypsinogen (24 kDa); Cyt C, Cytochrome-c (12.3 kDa). Lane 2, DEAE-Sepharose Fast Flow eluate (32.2 lg); Lane 3, Sephadex G-25 eluate (50.4 lg); Lanes 4–6, 2¢,5¢-ADP Sepharose 4B eluate: Lane 4, 3 lg; Lane 5, 1.5 lg; Lane 6, 0.75 lg.

et al., 1983; Tsai and Chen, 1975). In contrast, dimeric structures refer to higher subunit mass; e.g. for Corynebacterium glutamicum, Streptococus faecalis, Drosophila melanogaster, Bacillus stearothermophilus, Neurospora crassa and Haemophilus influenzae 6PGDs have dimeric structures with subunit masses from 50 to 70 kDa (Bianchi et al., 2001; Bridges et al., 1975; Pearse and Harris, 1973; Scott and Abramsky, 1973; Williamson et al., 1980; Yoon et al., 1989). The rat small intestine enzyme, like other mammalian 6-PDGs, is a dimer with subunit mass of 52 kDa (Fig. 3c).

3.2. Optimum pH Determination In the literature a wide range of pH optimum, (7 – 10) has been reported for 6PGD (Betts and Mayer, 1975; Beydemir et al., 2004; Bianchi et al., 2001; Bridges et al., 1975; Corpas et al., 1995; Cottreau and Boivin, 1975; Dyson et al., 1973; Pearse

and Harris, 1973; Pearse and Rosemeyer, 1974; Pearse and Rosemeyer, 1975; Procsal and Holten, 1972; Rosemeyer, 1987; Scott and Abramsky, 1973; Silverberg and Dalziel, 1973; Stournaras et al., 1983; Toews et al., 1976; Tsai and Chen, 1975; Villet and Dalziel, 1969; Weisz et al., 1985; Williamson et al., 1980). The reason for such a wide pH range may be that none of the studies used the zero buffer extrapolation method to eliminate the buffer effects. 6PGD from pig liver shows its maximal activity at pH 8.5 in glycine/NaOH, in tris/HCl and in bicarbonate buffers; in phosphate buffer the activity is constant from pH 7.5 to pH 10 (Toews et al., 1976). It was reported that 6PGDs isolated from other sources show a marked dependence on ionic strength (Dyson et al., 1973; Pearse and Rosemeyer, 1974; Procsal and Holten, 1972; Toews et al., 1976). The pH rate profile for the rat small intestinal 6PGD exhibited a wider pH optimum extending from 7.0 to 9.0 with increasing phosphate buffer concentration from 25 to 150 mM (not shown). Using data extrapolated to zero buffer extrapolation


299

yielded a sharper pH optimum profile and a value of 7.35 for rat small intestinal 6PGD was obtained (Fig. 4).

1.95

3.3. Determination of Ea, DH, Q10 and Optimum Temperature for 6PGD Although heat denaturation has been used as a purification step (Villet and Dalziel, 1969) reports on the heat stability of 6PGDs are limited. It was shown that 6PGDs isolated from sheep liver, rat erythrocytes, Bacillus stearothermophilus and Haemophilus influenzae had a temperature optimum of ‡45C (Dyson et al., 1973; Pearse and Harris, 1973; Yoon et al., 1989; Beydemir et al., 2004). The optimum temperature of 49.4C for 6PGD obtained from the rat small intestine well correlates with the values reported (Fig. 5). The enzymatic reaction had an activation energy (Ea) of 7.52 kcal/mol, activation enthalpy of 6.90 kcal/mol and Q10 of 1.49. Only one report was found in literature for the Ea, that was for sheep liver 6PGD. The reported value of 15.45 kcal/mol is twice what was found for the small intestinal 6PGD (Dyson et al., 1973) and this result may be explained by the difference in the origin of 6PGDs. 3.4. Steady-State Kinetics

0.65

0

-0.65 2.9

3.1

3.3 o

-1

(1/T) K x10

3.5

3

Fig. 5. Dependence of 6PGD Activity on Temperature. Preincubation (reaction) mixture: 100 mM phosphate buffer, pH 7.7, 10 mM MgCl2, 0.1 mM NADP were brought to desired temperature and the reaction was initiated by the addition of a suitable amount of the enzyme. (Vertical dotted line shows the optimum temperature for the rat small intestinal 6PGD).

range of the substrate, 6PG, 20–1280 lM and the coenzyme, NADP, 10–160 lM (Cleland, 1979). This is consistent with those determined for sheep liver 6PGD previously (Price and Cook, 1996) and contradictory with the result for the enzyme from pig liver (Toews et al., 1976). The Km values for 6PG and NADP were found to be 595±213 and 53.03±1.99 lM, respectively (Figs. 6 and 7).

8.0

0.20

1/v, U .mg protein

0.15

6.0

-1

Zero Buffer (KP) Extrapolation

The enzyme obeyed ‘‘Rapid Equilibrium Random Bi Bi’’ kinetic model in the concentration

Ln k

1.3

0.10

4.0

2.0

0.05 0.0 -0.01

0.00

0

0.01

0.02 -1

5.5

6.5

7.5

8.5

pH Fig. 4. Dependence of 6PGD Activity on pH. Phosphate buffers at different pH values and concentrations were used (All values extrapolated to zero buffer concentration).

1/[6-PGA], mM

Fig. 6. Lineweaver–Burk Plot for 6PG at constant [NADP]. The activities were determined at 37C in 100 mM phosphate buffer, pH 7.7, 10 mM MgCl2 and the reactions were initiated by the addition of the enzyme. [6PG], 40–640 lM; [NADP], (s) 10, (d) 20, (h) 40, (n) 80, (e) 160 lM.


300

1/v, U .mg protein

5.0

4.0

-1

3.0

2.0

1.0

0.0 -0.03

-0.01

0.01

0.03

0.05

-1

1/[NADP], µM

Fig. 7. Lineweaver–Burk Plot for NADP at constant [6PG]. The activities were determined at 37C in 100 mM phosphate buffer, pH 7.7, 10 mM MgCl2 and the reactions were initiated by the addition of the enzyme. [NADP], 10–160 lM; [6PG], (s) 40, (d) 80, (h) 160, (n) 320 and (e) 640 lM.

Secondary plots of 1/Vm versus 1/6PG (or 1/ NADP) yielded an intrinsic Vm value of 8.91±1.92 U/mg protein (Fig. 8). The Hill constants for 6PG and NADP were 1.13±0.15 and 0.89±13, respectively (Fig. 8 inset). The Km values reported for 6PG and NADP isolated from mammalian sources vary between 13.5 and 600 lM for 6PG and 3–258 lM for NADP (Procsal and Holten, 1972; Dyson et al., 1973; Betts and Mayer, 1975; Cottreau and Boivin, 1975; Pearse and Rosemeyer, 1975; Toews et al., 1976; Weisz et al., 1985;

-1

log(v / Vmax - v)

1 / Vm, U .mg protein

1.6

1.2

0.5 0.0 -0.5 -1.00.5

1.5

2.5

3.5

-1.5 -2.0

log[S]

0.8

0.4

0 0.00

0.02

0.04

0.06

0.08

0.10

-1

1/[6-PGA], 1/[NADP]; mM

Fig. 8. Double-Reciprocal Plots of Vm as a function of [6PG], (s) and [NADP], (d). Inset Hill plots for NADP, (s) and for 6PG, (d).

Rosemeyer, 1987; Corpas et al., 1995; Beydemir et al., 2004). In procaryotic 6PGDs these values are 23–102 lM for 6PG and 15–99 lM for NADP (Bridges et al., 1975; Tsai and Chen, 1975; Rosemeyer, 1987; Yoon et al., 1989; Bianchi et al., 2001). The kinetic parameters obtained for the rat small intestinal 6PGD are comparable with those obtained for 6PGDs from human granulocytes and rat erythrocytes (Cottreau and Boivin, 1975; Beydemir et al., 2004). Differences in the kinetic parameters of 6PGDs from different tissues of the same organisms were observed; i.e. the Km values of 6PGDs obtained from the rat liver and kidney cortex are reported as 257/49 lM for 6PG and 258/ 56 lM for NADP (Corpas et al., 1995). There are also differences between the kinetic parameters of the 6PGDs isolated from the same tissue by different research groups; the Km values reported for 6PG by Procsal et al. was 71 lM whereas Corpas reported a value of 157 lM and for NADP these values were 258 and 13 lM, respectively (Procsal and Holten, 1972; Corpas et al., 1995). The great variability observed in the Km values for 6PG and NADP for 6PGDs from mammalian and other sources might be mainly due to the different assay conditions and/or partly due to the origin of the enzyme. The enzyme, 6PGD, isolated from different tissues is inhibited by its coenzyme product, NADPH, competitively with respect to NADP (Procsal and Holten, 1972; Betts and Mayer, 1975; Cottreau and Boivin, 1975; Pearse and Rosemeyer, 1975; Weisz et al., 1985; Rosemeyer, 1987; Yoon et al., 1989; Corpas et al., 1995; Beydemir et al., 2004) and noncompetitively with respect to 6PG (Cottreau and Boivin, 1975; Weisz et al., 1985). The rat small intestinal 6PGD was competitively inhibited by its coenzyme product, NADPH, with a Ki of 31.91±1.31 lM (Fig. 9). The relatively small dissociation constant for 6PGD:NADPH complex pointed to tight enzyme:NADPH binding and the important role of NADPH in the regulation of the pentose phosphate pathway in vivo (Procsal and Holten, 1972). In toxicity investigations, rat is the most commonly used animal. The small intestine is an important route of entry for xenobiotics. Glutathione plays a central role in detoxification mechanisms. Characterization of 6PGD, together with the information on GSSGR, G6PD and GSTs from small intestine should allow assessment of the glutathi-


100 80

-1

1/Activity, U .mg protein

120

60 40

-Ki 20 0 -80

-40

0

40

80

120

160

NADPH, µM

Fig. 9. Dixon Plot for NADPH at different fixed [NADP] and at constant [6PG] = 600 lM. The activities were determined at 37C in 100 mM phosphate buffer, pH 7.7, 10 mM MgCl2 and the reactions were initiated by the addition of the enzyme (s) 20, (d) 40, (h) 80, (n) 120, (e) 200 and (r) 300 l M, [NADP].

one-related potential of this tissue (Tahir et al., 1988; O¨zer et al., 1990; Ogus and O¨zer, 1991; Danisan et al., 2004; Yildiz et al., 2004). It is clear that the contribution of 6PGD to the detoxification system in small intestine requires further investigation. The purification method presented above makes possible to obtain an at least 95 percent pure enzyme, which can be used in further studies.

REFERENCES Adams, M. J., Gover, S., Leaback, R., Phillips, C., and Somers, D. O’ N. (1991). Acta Cryst. 47: 817–820. Aksoy, Y., O¨gu¨s, I.H., and O¨zer, N. (2001). Prot. Exp. Purif. 21: 286–292. Betts, S. A., and Mayer, R. J. (1975). Biochem. J. 151: 263–270. Beutler, E. (1971). Red Cell Metabolism. New York and London: Grune & Stratton. Beydemir, S., Ciftci, M., Yilmaz, H., and Kufrevioglu, O. I. (2004). Turk J. Vet. Anim. Sci. 28: 707–714. Bianchi, D., Bertrand, O., Haupt, K., and Coello, N. (2001). Enzyme and Microbiol. Technology 28: 754–759. Bradford, M.M. (1976). Anal. Biochem. 72: 248–254. Bridges, R.B., Palumbo, M. P., and Wittenberger, C. L. (1975). J. Biol. Chem. 250: 6093–6100. Chasseaud, L. F. (1979). Adv. Cancer Res. 29: 175–274.

301

Cleland, W. W. (1979). Methods Enzymol. 63: 103–138. Corpas, F. J., Garcia-Salguero, L., Barroso, J. B., Aranda, F., and Lupianez, J. A. (1995). Mol. Cell. Biochem. 144: 97–104. Cottreau, D., and Boivin, P. (1975). Biochimie 57: 325–335. Danisan, A., Ceyhan, D., Ogus, I. H., and O¨zer, N. (2004). Protein J. 23: 317–324. Davies, B. J. (1964). Ann. N.Y.Acad. Sci. 121: 404–427. Dyson, J. E. D., D’Ozario, R. E., and Hanson, W. H. (1973). Arch. Biochem. Biophys. 154: 623–635. Eggleston, L. V., and Krebs, H. A. (1974). Biochem. J. 138: 425– 435. Krepinsky, K., Plaumann, M., Martin, W., and Schnarrenberger, C. (2001). Eur. J. Biochem. 268: 2678–2686. Laeemli, U. K. (1970). Nature 227: 680–685. Levy, H. R. (1979). Adv. Enzymol. Rel. Areas Mol. Biol. 48: 97–192. Meister, A., and Anderson, M. E. (1981). Ann. Rev. Biochem. 52: 711–760. Ogus, I. H., and O¨zer, N. (1991). Biochem. Med. Met. Biol. 45: 65– 73. O¨zer, N., Aksoy, Y., and O¨gu¨s, I. H. (2001). Int. J. Biochem. Cell Biol. 33: 221–226. O¨zer, N., Bilgi, C., and O¨gu¨s, I. H. (2002). Int. J. Biochem. Cell Biol. 34: 253–262. O¨zer, N., Erdemli, O¨., Sayek, I., and O¨zer, I. (1990). Biochem. Med. Met. Biol. 44: 142–150. Pearse, B.M.F., and Harris, J. I. (1973). FEBS Lett. 38: 49–52. Pearse, B. M. F., and Rosemeyer, M. A. (1974). Eur. J. Biochem. 42: 213–223. Pearse, B.M.F., and Rosemeyer, M. A. (1975). Methods Enzymol. XLI: 220–226. Price, N. E., and Cook, P. F. (1996). Arch. Biochem. Biophys. 336: 215–223. Procsal, D., and Holten, D. (1972). Biochem. 11: 1310–1314. Rippa, M., Hanau, S., Cervellati, C., and Dallocchio, F. (1998). Prot. Pept. Lett. 7: 341–348. Rosemeyer, M. A. (1987). Cell. Biochem. Func. 5: 79–95. Scott, W. A., and Abramsky, T. (1973). J. Biol. Chem. 248: 3535– 3541. Segel, I. H. (1975). Enzyme Kinetics. U.S.A. John Wiley & Sons, Inc. Silverberg, M., and Dalziel, K. (1973). Eur. J. Biochem. 38: 229– 238. Stournaras, C., Maurer, P., and Kurz, G. (1983). Eur. J. Biochem. 130: 391–396. Tahir, M. K., O¨zer, N., and Mannervik, B. (1988). Biochem. J. 253: 759–764. Toews, M. L., Kanji, M. I., and Carper, W. R. (1976). J. Biol. Chem. 251: 7127–7131. Tsai, C. S., and Chen, Q. (1998). Biochem. Cell Biol. 76: 637–644. Villet, R. H., and Dalziel, K. (1969). Biochem. J. 115: 639–643. Vuopio, P., Ha¨rko¨ne¨n, M., Johnson, R., and Nuutinen, M. (1973). Ann. Clin. Res. 5: 168–173. Weisz, K. S., Schofield, P. J., and Edwards, M. R. (1985). J. Neurochem. 44: 510–517. Williamson, J.H., Krochko, D., and Geer, B. W. (1980). Biochem. Genet. 18: 87–101. Wilson, J. E. (1971). Arch. Biochem. Biophys. 147: 471–474. Yildiz, O¨., Bodur, E., Cokugras, A. N., and O¨zer, N. (2004). Protein J. 23: 143–151. Yoon, H., Anderson, C. D., and Anderson, B. M. (1989). Biochem. Biophys. Acta 994: 75–80. Yoshida, A. (1966). J. Biol. Chem. 241: 4966–4976.