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Enzymic Conversion of 3-Hydroxyanthranilic Acid Into Cinnabarinic Acid PARTIAL PURIFICATION and PROPERTIES of RAT-LIVER CINNABARINATE SYNTHASE by P

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Enzymic Conversion of 3-Hydroxyanthranilic Acid Into Cinnabarinic Acid PARTIAL PURIFICATION and PROPERTIES of RAT-LIVER CINNABARINATE SYNTHASE by P Biochem. J. (1966) 99, 317 317 Enzymic Conversion of 3-Hydroxyanthranilic Acid into Cinnabarinic Acid PARTIAL PURIFICATION AND PROPERTIES OF RAT-LIVER CINNABARINATE SYNTHASE By P. V. SUBBA RAO AND C. S. VAIDYANATHAN Department of Biochemistry, Indian Institute ofScience, Bangalore, India (Received 20 October 1965) Rat-liver cinnabarinate synthase (3-hydroxyanthranilic acid-oxygen oxido- reductase) was partially purified. Stoicheiometric studies indicated the con- sumption of 3 atoms of oxygen/molecule of cinnabarinic acid formed. There was an initial lag in enzyme activity. The reaction had an optimum pH about 7-2 and an optimum temperature of 37°. The enzyme was highly specific for 3-hydroxyanthranilic acid. The system showed an absolute requirement for Mn2+ ions. Several bivalent metal ions and metal-chelating agents inhibited the reaction. Thiol inhibitors had no effect on enzyme activity, but reducing agents such as ascorbic acid were potent inhibitors. There was no requirement for any cofactor other than Mn2+ ions. The probable significance of the reaction in mammals is discussed. Recent interest in the initiation of bladder tumours in experimental animals (Bryan, Brown & MATERIALS AND METHODS Price, 1964) by certain o-aminophenols such as All reagents were of analytical grade and glass-distilled 3-hydroxyanthranilic acid and 3-hydroxykynure- water was used for the preparation of all solutions. nine, which are normal metabolites, as well as the Chemical&. 3-Hydroxyanthranilic acid was purchased excretion of abnormally large quantities of trypto- from Mann Research Laboratories Inc., New York, N.Y., U.S.A. Horse-heart cytochrome c, horse-radish peroxidase, phan metabolites in the urine of patients bearing ox-liver catalase, NAD, NADP, FMN, FAD and p-chloro- bladder tumours in a normal population (Boyland mercuribenzoate were obtained from Sigma Chemical Co., & Williams, 1956; Brown, Price, Satter & Wear, St Louis, Mo., U.S.A. Sodium arsenite, 2,3-dimercapto- 1960), calls for a fresh search for alternative path- propanol, cysteine and GSH were obtained from British ways for the metabolism of these tryptophan Drug Houses Ltd., Poole, Dorset. Haem and haematin metabolites. The enzymic conversion of 3-hydroxy- were prepared from Sigma haemin. anthranilic acid into cinnabarinic acid (2-amino- Purification ofenzyme. All operations were carried out at 3-oxo-3H-phenoxazine-1,9-dicarboxylic acid) by 0-4o. the nuclear fraction of rat liver has been described Step 1. The nuclear fraction from 25g. of rat liver was isolated according to the procedure of Subba Rao, by Subba Rao, Jegannathan & Vaidyanathan Jegannathan & Vaidyanathan (1965). The nuclear fraction (1964). It was reported by Morgan & Weimorts obtained from 25g. of liver was finally suspended in 65ml. (1964) that acetone-dried powders from livers of of 0.9% NaCl. poikilothermic animals can also catalyse such an Step 2. The nuclear fraction (65ml.) was centrifuged at enzymic oxidation of 3-hydroxyanthranilic acid. 12000g for 5min., the sediment was suspended in 60ml. This enzyme, which has been purified by Morgan, of O-OlM-sodium phosphate buffer, pH7-0, and homo- Weimorts & Aubert (1965), is unspecific and can genized in a VirTis homogenizer for 5-lOmin. The extract oxidize related o-aminophenols also to phenoxazine was centrifuged at 12000g for lOmin. compounds. In view of the carcinogenic activity Step 3. To 54ml. of solubilized extract from step 2 were further studies added 6ml. of 0-lM-MnSO4. The mixture was stirred for ofcinnabarinic acid (Boyland, 1960), 15min. and centrifuged at 12000g for 15min. The precipi- on the enzyme cinnabarinate synthase have been tate was discarded and the clear supernatant was dialysed carried out. The enzyme from rat liver has been thoroughly against water and clarified by centrifugation. partially purified and properties of this enzyme, Step 4. The fraction from step 3 (50nml.) was brought to which is specific for 3-hydroxyanthranilic acid, are 30% saturation by the addition of 8-2g. of recrystallized described. (NH4)2SO4, stirred for lBmin. and centrifuged at 12000 g 318 P. V. SUBBA RAO AND C. S. VAIDYANATHAN 1966 Table 1. Purification of cinnabarinate synthase from rat liver The reaction mixture (1 ml.) containing sodium phosphate buffer, pH7*2 (50,umoles), 3-hydroxyanthranilic acid (4,umoles), MnSO4 (0.25/umole) and O lml. of enzyme was incubated at 370 for 30min. (Preparations from steps 1-5 were diluted tenfold and the final preparation was diluted fivefold.) Cinnabarinic acid formed in the reaction mixture was estimated as described in the Materials and Methods section. A unit of activity is defined as the amount of enzyme that catalyses the formation of 1,mole of cinnabarinic acid/min. Specific Total Total activity Volume protein activity (units/mg. Recovery Step (ml.) (mg.) (units) of protein) (%) Purification 1 65 845 112-2 0-13 100 2 60 366 66-0 0-18 58-9 1.4 3 62 149 59.5 0*41 53-1 3*2 4 41 31-2 33-2 1-06 29-6 8-2 5 27 6*75 22-5 3-30 20-0 25-4 6 54 2-38 20-3 8*53 18.1 65-6 for 15min. The precipitate was discarded. The supernatant 0 4 was brought to 60% saturation by the addition of 9 59g. of (NH4)2SO4. After stirring for 15min., the precipitate was collected by centrifugation, suspended in 33ml. of water and dialysed against water. Step 5. The dialysed preparation (step 4) (30ml.) was fractionated at -4° with acetone precooled to -20°. The 0L2 precipitate obtained at 15-40% (v/v) acetone was collected, E dissolved in 20ml. of water and centrifuged to get a clear solution. 01I Step 6. The solution (15ml.) of the precipitate from the acetone fractionation was adjusted to pH6*0 with dilute acetic acid, and tricalcium phosphate gel (45 ml. of a 0 preparation containing 14mg./ml.; Keilin & Hartree, 1938) 340 380 420 460 500 was added. The mixture was stirred for 15min. and the Wavelength (mu) gel was separated by centrifugation and resuspended in 30ml. of 005m-sodium phosphate buffer, pH7-2. After Fig. 1. Visible spectrum of cinnabarinic acid (6.2tLg./ml.) 20min. the mixture was centrifuged and the clear solution in an aqueous solution of 1% (w/v) trichloroacetic acid. was used as enzyme. The results of the purification procedure are given in Table 1. Measurement of enzyme activity. The spectrum of cinna- Oxygen uptake. Warburg manometers contained sodium barinic acid in 1% (w/v) trichloroacetic acid as taken on a phosphate buffer, pH7.2(1001&moles), 3-hydroxyanthranilic Beckman model DB recording spectrophotometer is shown acid (6,umoles), MnSO4 (0.75umole) and 1 ml. of enzyme in Fig. 1. The absorption maxima were at 430mit and in a final volume of 3ml. The reaction was started by the, 450-452m,u. The molar extinction coefficient (e) at 450mi. addition of substrate from the side arm and the oxygen was 17280, and there was linearity between the extinction uptake was measured over a period of 60min. at 300. at 450m,u and concentration of cinnabarinic acid in the Spectrophotometry. The reduction and reoxidation of range 5-40,tg./3 ml. cytochrome c in the presence of 3-hydroxyanthranilic acid The standard reaction mixture consisted of sodium and the enzyme was measured in cuvettes maintained at phosphate buffer, pH7.2 (50,tmoles), 3-hydroxyanthranilic 30°. Difference spectra were recorded on a Beckman model acid (4,umoles), MnSO4 (0 25,umole) and Ol ml. of enzyme DB recording spectrophotometer. The reaction mixture (0.8jg. ofprotein) in a totalvolume of1 ml. After incubation (3.Oml.) consisted of sodium phosphate buffer, pH7.2 for 30min. at 37°, the reaction was stopped by the addition (100l,moles), cytochrome c (0-2,umole), MnSO4 (0 3,umole) of 1 ml. of 3% (w/v) trichloroacetic acid followed by 1 ml. and 3-hydroxyanthranilic acid (0.1 imole). It was scanned of water. After centrifugation, 1 ml. samples were diluted against a blank in which the substrate was omitted. After to 3ml. with 1% (w/v) trichloroacetic acid, and cinnabarinic the addition of 0-5ml. of enzyme to both cuvettes, the acid formed was calculated from the extinction at 450m,u. spectrum was recorded at 5min. intervals. To correct for non-enzymic oxidation of3-hydroxyanthran- flic acid, blanks containing boiled enzyme were set up in RESULTS each experiment. Determination ofprotein. This was done according to the Requirementfor Mn2+ ions. The partially purified method of Lowry, Rosebrough, Farr & Randall (1951). enzyme showed an absolute requirement for Mn2+ Vol. 99 RAT-LIVER CINNABARINATE SYNTHASE 319 Table 2. Effect of Mn2+ ions on cinnabarinic acid 0 5 formation by purified rat-liver cinnabarinate synthase a) Enzyme containing 0-8j,g. of protein was preincubated 0 with the indicated final concentration of MnSO4 for 5min. 0-4 at 370 before the substrate was added. The rest of the procedure was as described in the Materials and Methods 0) section. 0- 3 .0 Concn. of MnSO4 Cinnabarinic acid formed IC (mM) (,umole) 0 o 0 2 - 0 9: 0-01 0-05 .; I 0-025 0-09 Ca0*Ca 0-05 0-12 0-1 _ 0-1 0-15 0 0-25 0-18 0-5 0-12 120 150 1-0 0-10 0 30 60 90 Time (min.) Fig. 2. Time-course of cinnabarinic acid formation in the presence of purified rat-liver cinnabarinate synthase. The ions, the optimum concentration of which was reaction mixture consisted of sodium phosphate buffer 0-25mm in the presence of 4mM-3-hydroxy- (50umoles), 3-hydroxyanthranilic acid (4,umoles), acid MnSO4 anthranilic (Table 2). None of the other (0-25,umole) and enzyme (0-8,g. of protein) in a final bivalent metal ions Cu2+, Fe2+, Ca2+, Mg2+ and volume of lml.
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