Organ distribution, purification and characterization of kynureninase in Suncus murinus (Insectivora) and anthranilic acid level in the serum

Comp. Biochem. Physiol. Vol. 93B, No. 1, pp. 107-111, 1989 0305-0491/89 $3.00 + 0.00 Printed in Great Britain © 1989 Pergamon Press plc ORGAN DISTRIBUTION, PURIFICATION AND CHARACTERIZATION OF KYNURENINASE IN SUNCUS MURINUS (INSECTIVORA) AND ANTHRANILIC ACID LEVEL IN THE SERUM TAKAYA ISHIKAWA, ETSOO OKUNO, JUN KAWAI and RYO KIDO* Department of Biochemistry, Wakayama Medical College, 27 Kyubancho, Wakayama 640, Japan (Tel: 734 31 2151) (Received 27 June 1988) Abstract--1. The highest kynureninase activity was measured in Suncus liver compared with other organs tested. The holo-enzyme activity detected in liver was 25%. 2. The enzyme mainly localized in liver cytosol was purified to a single protein band by heat treatment, ammonium sulfate fractionation, DEAE-sepharose, hydroxyapatite and phenyl-sepharose column chromatography. 3. The purified enzyme was effective for both kynurenine and 3-hydroxykynurenine, and showed the optimum pH at 8.5 against both substrates. 4. Km values were 250#M, 18 #M and 70#M for kynurenine, 3-hydroxykynurenine and pyridoxal Y-phosphate, respectively. Various compounds such as histidine, aspartate and nicotinamide enhanced the enzyme activities. 5. Anthranilate, the product of kynureninase, was present at a concentration in Suncus serum of 1.96/~M. This value was higher than those of rat and human. INTRODUCTION The enzyme kynureninase (L-kynurenine hydrolase, EC 3.7.1.3.) catalyzes the hydrolysis of both L- kynurenine (yielding anthranilate) and L-3-hydroxy- kynurenine (yielding 3-hydroxyanthranilate). The latter is an intermediate which is on the physiologi- cally important pathway to nicotinamide nucleotides from tryptophan (Bender and McCreanor, 1985). Braunstein et aL (1949) demonstrated a decrease of the kynureninase activity on pyridoxine deficiency, showing that the enzyme requires pyridoxal 5'-phos- phate as a coenzyme. Several other workers have studied the effect of pyridoxine deficiency on trypto- phan metabolism, particularly on the kynureninase activity. In regard to organ distribution, rat liver contains high kynureninase activity, while the kidney and spleen have lower activity and the activity in intestine, heart, lung, adrenal gland, muscle and brain is small (Kawai et al., 1988). Other mammalian livers possess high kynureninase activity and the livers of other vertebrates, i.e. birds, reptiles, amphibia and fishes also show kynureninase activity (Gaertner and Shetty, 1977), suggesting that the enzyme is in- dispensable in animal livers. We found an extremely high activity of kynurenin- ase in Suncus liver. The present paper describes the properties of purified kynureninase and anthranilate level in the serum. *Author to whom correspondence should be addressed. MATERIALS AND METHODS Animals Male Suncus (Suncus murinus, 8 weeks old, 120-130g body wt) were provided by our animal breeding room. They were maintained at about 20°C in a room with a 12 hr light/12 hr dark cycle. Food (MF laboratory chow, Oriental Yeast Co. Ltd, Tokyo, Japan) and water were available ad libitum. Chemicals L-Kynurenine was prepared by ozonolysis of L-trypto- phan by the method of Wamell and Berg (1954). 3- Hydroxy-L-kynurenine was purchased from Wako Pure Chemicals Ltd, Tokyo, Japan. Anthranilic acid was ob- tained from Katayama Chem. Co. Ltd, Osaka, Japan and recrystallized from water after treating it with activated charcoal. Pyridoxal Y-phosphate and 3-hydroxyanthranilic acid were purchased from Nakarai Chemicals, Ltd, Kyoto, Japan. Sephadex G-25, DEAE-Sepharose and Phenyl- Sepharose were from Pharmacia Fine Chemicals, Uppsala, Sweden. Amphonine carrier ampholite was from LKB- Producter AB, Stockholm, Sweden. All other chemicals used were commercial products of the highest grade available. Enzyme assays The kynureninase activities against kynurenine or 3- hydroxykynurenine were assayed under conditions giving linear progress curves and where the initial rate was propor- tional to enzyme concentration. Anthranilate of 3-hydroxy- anthranilate produced was measured fluorometrically as described by Shetty and Gaertner (1973). The standard assay mixture (100#1) contained 100mM of Tris-HC1 buffer, pH8.5, 2mM of kynurenine or 0.2mM of 3- hydroxykynurenine, 0.1 mM of pyridoxal Y-phosphate and 107 108 TAKAYA ISHIKAWA et al. enzyme solution. A unit of enzyme activity is defined as the amount of enzyme catalyzing the formation of 1/amol of anthranilate of 3-hydroxyanthranilate per min at 3T'C. Anthranilic acid assay Suncus and rat blood were obtained by heart puncture. Human blood samples were collected from four healthy males (age range 3540 years). Blood cells were separated by centrifugation (1000 g, 10 min). The supernatant serum was frozen at -2ff 'C until analysis. After thawing, 1 ml of serum was added to 0.1 M HCI and boiled for 5 min and precipi- tated protein was separated by centrifugation. The resulting supernatant was applied to a column (0.5 × 1.5cm) of Dowex 50 H + form. After washing with I0 ml of 0.1 M HC1 and 30 ml of H20, anthranilic acid was eluted with 10 ml of 1 N NH4OH. The eluate was dried by EYELA vapor mix S-10 (Tokyo Rikakikai Co. Ltd, Tokyo, Japan) in vacuo at 30'C. Anthranilic acid was extracted and dissolved with 100/al of 0.5 N NaOH. Anthranilic acid was detected by an HPLC system with a fluorometric detector as described before (Kawai et al., 1988). As a standard 5nmol of anthranilic acid was treated with the same method. The recovery rate was up to 95% from boiling and column. Thirty per cent of anthranilic acid however was lost in the drying step. Other methods Molecular weight determination by sucrose density gradient centrifugation, isoelectric focusing and protein measurement were carried out as described before (Kawai et al., 1988). Purification of kynureninase fi'om Suncus liver All manipulations were performed at 0.4'C and glass- distilled water and potassium phosphate buffer, pH 7.5 containing 2 mM mercaptoethanol and 200/aM pyridoxal 5'-phosphate were used throughout unless stated otherwise. Two animals (body wt l10-120g) were decapitated. The livers (5.0 g) were removed immediately and homogenized with 9 vol of 0.15 M potassium chloride containing 2 mM mercaptoethanol and 200/~M pyridoxal 5'-phosphate in a Potter-Elvehjem homogenizer with a Teflon pestle. After centrifugation at 10,000 g for 30 min of the homogenate, the precipitate was discarded and the resulting supernatant was heat-treated at 65°C for 1 min and immediately cooled to 4'C in an ice bath. The heat-denatured protein was removed by centrifugation. Solid (NH4)2SO 4 was added to the heat-treated enzyme solution, with gentle stirring, to 30% saturation. After 30 min, the precipitate was removed by centrifugation at 10,000 g for 20 min and discarded. (NH4)~SO4 was added to the supernatant to 75% saturation, and after 30 min, the precipitate was collected by centrifugation at 10,000g for 20 min. The precipitate was dissolved in a minimum volume of 5 mM buffer and the enzyme solution was desalted by the use of a Sephadex G-25 column which had been equilibrated with 5 mM buffer. The resulting enzyme solution was applied to a column (2.5 × 5 cm) of DEAE-Sepharose equilibrated with 5 mM buffer. Pyridoxal-phosphate was omitted in this step. The column was washed with 100 ml of 5 mM buffer and the enzyme was eluted with a linear gradient from 5-250 mM buffer with a total volume of 600 ml. Active fractions were combined and concentrated by (NH4)2SO4 to 75% satur- ation. After centrifugation, the precipitate was dissolved in a minimum volume of 5 mM buffer containing pyridoxal- phosphate and desalted by the use of a Sephadex G-25 column equilibrated with the same buffer. The enzyme solution was injected into a column (1.5 x 5 cm) of hydroxyapatite which was equilibrated with 5 mM buffer. After washing the column with 50 ml of 5 mM buffer, the enzyme was eluted with a linear gradient from 5 250mM buffer with a total volume of 400ml. Active fractions were pooled together and (NH 4 )2 SO4 was added to 20% saturation. The enzyme solution in 20% ammonium sulfate was applied to a column (1.0 × 7cm) of Phenyl-Sepharose which was equilibrated with 20% ammonium sulfate. The column was washed with 50 ml of 20% ammonium sulfate and the enzyme was eluted with a linear gradient from 20% ammonium sulfate to 200 pM of pyridoxal-phosphate and 2 mM mercaptoethanol. The active fractions were pooled and concentrated by collodion bag in vacuo and dialysed against 1000 ml of 5 mM buffer. Preparation of apo-enzyme The apo-enzyme was prepared by incubation of the holo-enzyme with 5 mM phenylhydrazine in 50 mM potas- sium phosphate buffer, pH 7.5 at room temperature for 30 min, followed by gel filtration on Sephadex G-25 equili- brated with the same buffer. This procedure was repeated twice. The apo-enzyme had less than 0.5% residual activity. RESULTS Organ distribution Four male Suncus (body weight 120-130 g) were killed by decapitation after having been anesthetized by ether. The organs were removed quickly and homogenized in a Potter-Elvehjem homogenizer with a Teflon pestle with 5 vol of 0.25 M sucrose. The organ distribution of kynureninase was investigated by measuring the enzyme activity. The highest ac- tivity was found in the liver, of which holo-enzyme was 25%, while other organs tested (kidney, thymus, spleen, intestine, heart, lung, brain and muscle) showed less than 1/300 of liver activity (Table l). All kynureninase in brain and muscle, which showed lower activities than other organs, existed as holo-enzyme. Criteria o f purity and molecular weight of the enzyme The kynureninase from Suncus liver was purified as written in the "Methods" section. Results of a typical purification are shown in Table 2. The enzyme was purified about 65-fold over the Suncus liver homogenate, giving 18% yield. On polyacrylamide-disc-gel electrophoresis at pH 8.9 in 7% gel, the purified enzyme migrated toward the anode with mobility of 0.30 relative to Bromophenol Blue (the tracking dye) as a single protein band, which coincided with kynureninase activity (data not shown). Also the purified enzyme was subjected to electrophoresis on a polyacrylamide- slab-gel containing sodium dodecyl sulfate, giving a Table 1. Organ distribution of kynureninase in Suncus liver Kynureninase activity (uU/mg protein) % Of Organ with PALP without PALP holo-enzyme Liver 17,300 + 2100 4410 + 1060 25 Kidney 47.1 ± 7,6 32.3 + 5.9 69 Thymus 31.8 _+ 2.9 8.2 _+ t.8 26 Spleen 24.7 ± 2.3 4.7 ± 0.6 19 Small intestine 11.2 ± 2.9 5.9 + | .2 53 Heart 7.6 ± 2.4 1.8 + 0.6 24 Lung 5.3 + 0.6 1.8 ± 0.5 34 Whole brain 2.9 ± 0.4 2.9 ± 0.4 100 Muscle 1.8 _+ 0.3 1.8 + 0.3 100 Data are the means _+ SEM of four separate experiments. PALP: pyridoxal 5'-phosphate. Kynureninase in a shrew Table 2. Purification of kynureninase from Suncus liver Total Total Specific protein activity activity Recovery Purification (mg) (U) (U/mg) (%) (fold) Homogenate 887 15.4 0.017 100 1 Supernatant 557 13.8 0.024 90 1.4 Heat 169 12.9 0.076 84 4.5 (NH4)2 SO4 90.6 11.3 0.125 73 7.4 DEAE Sepharose 19.9 8.46 0.425 55 25 Hydroxyapatite 12.0 6.78 0.565 44 33 Phe-Sepharose 2.5 2.75 1.10 18 65 109 single protein band that had an estimated tool. wt of 56,000 (Fig. 1). The mol. wt was estimated as 110,000 by sucrose density gradient centrifugation, showing that this enzyme probably consists of two identical subunits. Isoelectric point A typicat isoelectric focusing profile of whole homogenate extract from liver is shown in Fig. 2. One peak of kynureninase activities with kynurenine and 3-hydroxykynurenine was obtained with the identical profile, possessing the same isoelectric point pH 6.4 (Fig. 2). The activity ratio of the enzyme towards 3-hydroxykynurenine and kynurenine was about 1.5. This ratio was the same as for the purified enzyme. Kinetic properties Kinetic properties for both substrate, kynurenine and 3-hydroxykynurenine may help the consideration of their role in metabolism. The apparent K,~ values were determined from double reciprocal plots of initial velocity and substrate concentration. Apparent Km values were 18#M for 3-hydroxykynurenine and 250FM for kynurenine (Fig. 3(a),(b)). The Km value for pyridoxal Y-phosphate by using Origin 4 3 2 1 2 Fig. 1. Sodium dodecylsulfate polycrylamide gel electro- phoresis of purified enzyme. The gel was stained with Coomassie Blue. Lane 1, standard proteins, from top to bottom ct:-macroglobulin from human plasma, fl- galactosidase from Escherichia coli, fructose-6-phosphate kinase from rabbit muscle, pyruvate kinase from chicken muscle fumarase from porcine heart, lactic dehydrogenase from rabbit muscle and triosephosphate isomerase from rabbit muscle. Lane 2, purified kynureninase from Suncus liver. apo-enzyme was 70#M with kynurenine for substrate (Fig. 3(c). pH-Dependence Over the pH range 7.6-9.5 purified kynureninase showed nearly identical activity profiles with kynurenine and 3-hydroxykynurenine as substrate with pH optimum at 8.5 (Fig. 4). Effect of various compound on the activity Various amino acids or kynurenine metabolites were added in the reaction mixture of kynureninase assay with kynurenine or 3-hydroxykynurenine for substrate. Only L-alanine inhibited the enzyme ac- tivity with kynurenine 14%. The enzyme activity with 3-hydroxykynurenine however was enhanced a little by addition of L-alanine. All other compounds tested enhanced both the enzyme activity with kynurenine and 3-hydroxykynurenine. In particular, enzyme ac- tivity with kynurenine was enhanced by the addition of histidine and enzyme activity with 3-hydroxy- kynurenine was enhanced by histidine and aspargine (Table 2). Double reciprocal plots revealed the mech- anism of activation for kynureninase with kynurenine and 3-hydroxykynurenine by addition of histidine (50 mM) that depend on increasing of both Km value and lima x (Fig. 3(a),(b)). Relationship of kynureninase activity and anthranilic acid level in the serum Suncus liver contained about a hundred times and a thousand times higher specific activity than rat liver 15G "" . , , . " I0 i 10( 10 20 30 Fraction number Fig. 2. Isoelectric focusing of the enzyme from Suncus liver. The supernatant obtained after centrifugation at 100,000g for 30min (0.1 g of original tissue) from liver which was homogenized with 9 vol of 0.25 M sucrose by Potter- Elvehjem homogenizer, was subjected to isoelectric focus- ing. Fractions of 2.5 ml were collected, pH values ( x ) and kynureninase activities 3-hydroxykynurenine (O) and kynurenine (O) for substrate were determined as described in the text. Values of a typical experiment are reported. l l0 TAKAYA ISHIKAWA el al. a) ~... 50 25 0 . . . . i i 2.5 _~ ,5 I / l Nyn](mM) o i , j 0 25 -150 1/[3-0H Kynl(mM) c) 6O v ~4o & / 1 0.25 0.5 1 / [PALP](,,uM)-' Fig. 3. Double reciprocal plots of initial velocity of purified kynureninase with kynurenine (a) and 3-hydroxykynurenine (b) for substrate in the presence of 50 mM (@) or absence ((3) of histidine. Double reciprocal plots of the enzyme against pyridoxal 5"-phosphate (c) with kynurenine for substrate. and human liver, respectively. Anthranilic acid level in the Suncus serum however was about 8 times and 5 times greater than those in rat and human, respectively. DISCUSSION Kynureninase as a key enzyme on the NAD path- ways in tryptophan metabolism, catalyzes a unique reaction, the hydrolytic fl,7 cleavage of kynurenine or 3-hydroxykynurenine. Suncus liver contains higher kynureninase activity than those of other mammalian livers, showing activity ratios of about 50 times that of pig liver (Tanizawa and Soda, 1979), 100 times that of rat liver (Kawai et al., 1988) and 1000 times that of human liver (Inada et al., 1984). Percent of holo- enzyme in Suncus liver and kidney were 25% and 69%, respectively. That of rat liver is about 50% (Rose and Brown, 1969). Those values might reflect the K,. value for pyridoxal 5'-phosphate (Suncus; 79#M and rat; 0.8#M) and concentration of enzyme. The enzyme in suncus liver was purified to homogeneity from only 5 g of liver. Purified Suncus kynureninase has a lower K,, value for 3-hydroxy- kynurenine than kynurenine. This characteristic is similar to other mammalian liver kynureninases (Tanizawa and Soda, 1979; Takeuchi et al., 1980). Addition of amino acids showed inhibitory or no 150 I ,oo 50 I . . . . 1 7.5 8.0 8,5 9,~3 915 pH Fig. 4. Effect of pH on purified kynareninase with kynurenine ((3) and 3-hydroxykynurenine (@). Assay con- ditions were as described in the Methods section, except that Tris HCI buffer was at pH 7.6-9.5. effect on rat kynureninase (Haglund et al., 1977) and kynureninase of Presudomonas marginalis (Moriguchi et al., 1973). In regard to Suncus kynureninase how- ever, only e-alanine showed inhibitory effect on kynureninase activity and all other compounds tested showed activatory effect on both kynureninase and 3-hydroxykynureninase activity (Table 3). The relationship between kynureninase activity and anthranilate level in the serum is examined in Table 4. The kinetic analysis of the liver kynureninase shows that 3-hydroxykynurenine is the best substrate. Tanizawa and Soda (1979) have reported that 3-hydroxykynurenine is probably the physiological substrate of the enzyme. However we found signifi- cant amounts of anthranilic acid in the serum. Higher anthranilic acid level in the serum was observed in Suncus with higher kynureninase activity (Table 4). As regards anthranilic acid concentration in human serum, it was higher than that of rat. It is possible to reason that kynureninase activity in the human liver is decreased by treatment with anti-cancer drugs (Inada et al., 1984). There are three directions of kynurenine metabolism, these are: cleavage to anthranilic acid and alanine by kynureninase, transamination by kynurenine aminotransferase, and hydroxylation by kynurenine 3-hydroxylase. K,, values for kynurenine of kynurenine 3-hydroxylase in rat liver (Bender and McCreanor, 1982), kynurenine aminotransferase in rat kidney (Noguchi and Kido, 1976) and human Table 3. Effects of various compounds (5 mM) on kynureninase Relative aclivity for Compound_ . . . . . K ynureniue 3-Hydroxykynurenine None 100 100 L-Alanine 86 I 15 D-Alanine 123 124 Nicotinate 137 119 Nicotinamide 125 107 Histidine 149 143 Aspartate 121 I 17 Asparagine 133 149 Quinolinate 130 115 Kynureninase in a shrew 111 Table 4. Kynureninase activity and anthranilic acid in rat, Suncus and human Kynureninase Anthranilic activity acid in serum (nmol/min/mg) (#M) Rat 0.21 + 0.04* 0.25 + 0.02 Suncus 25.95 + 6.94 1.96 + 0.24 Human 0.028t 0.42 + 0.03 *From Kawai et al. (1988). tFrom Inada et al. (1984). liver (Okuno et al., 1980) and kynureninase in rat liver (Takeuchi et al., 1980) have been reported to be 18/zM, 1.4 mM, 5.0 mM and 240 #M, respectively. From these Km values, kynurenine metabolism is probably by way of hydroxylation. However there are many reports that the hydroxylation of kynurenine may be rate-limiting on the basis of measurement of the urinary excretion of kynurenine and 3-hydroxy- kynurenine (Rose and McGinty, 1968; Musajo and Benassi, 1964; Michael et al., 1964). 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