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Properties of a Newenzyme,Nucleoside Oxidase

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Properties of a Newenzyme,Nucleoside Oxidase Agric. Biol. Chem., 53 (6), 1671 -1677, 1989 1671 Properties of a NewEnzyme, Nucleoside Oxidase, from Pseudomonas maltophilia LB-86 Yoshikazu Isono, TomokoSudo and MasamiHoshino Biwako Research Laboratory, Otsuka Foods Co., Ltd., Karasaki, Otsu, Shiga 520-01, Japan Received January 23, 1989 Nucleoside oxidase purified from Pseudomonas maltophilia LB-86 had mol. wt. = 130.000 and was composed of one each of four non-identical subunits: subunit a, 76,000; subunit /?, 33,000; subunit y, 18,000; subunit <5, 14,000. The enzyme contains 1 mol of covalently bound FAD, 2g atoms of non- heme iron, 2 mol of labile sulfides, and 1 mol of heme per mol enzyme protein. The absorption spectrum of nucleoside oxidase had maxima 278 and 390nm, and shoulders at 343 and 450nm. The enzymecatalyzes the oxidation of various nucleosides, and the Kmvalue for inosine was 4.4 x 10"5 M. The enzyme was most active at pH 5-6, and was most stable between pH 5.0-6.0 and at temperatures below 60°C. The activity was strongly inhibited by /V-bromosuccinimide and potassium cyanide. As reported previously,1} a new enzyme oxidase was prepared from Pseudomonas maltophilia LB- catalyzed the oxidation of nucleosides without 86, as described previously.2' a requirement for an exogenous co factor. This nucleoside-oxidizing enzyme was quite dif- Assay of nucleoside oxidase activity. Enzymeactivity was assayed by measurementof oxygen uptake with an ferent from any previously reported enzymes, Oxygraph model 8 oxygen electrode (Central Kagaku Co., and thus we have designated tentatively it as Ltd.) placed in a thermostatically controlled vessel at "nucleoside oxidase." Nucleoside oxidase was 25°C. The reaction mixture contained 7jumol of inosine, 100/miol of potassium phosphate buffer (pH 6.0), and a purified from the cell-extract of Pseudomonas suitable amount of nucleoside oxidase in a final volume of maltophilia LB-86 by ammoniumsulfate frac- 1.0ml. One unit of enzyme activity was defined as the tionation, heat treatment, columnchramatog- amount of enzyme that consume 1jimo\ of oxygen raphy on DEAE-Toyopearl, and gel filtration per min. twice on a Sephacry S-200 column.2) The purified enzyme preparation was homo- Measurement of protein. Protein was measured by the geneous on polyacrylamide gel electro- method of Lowry et al.,3) with crystalline bovine serum albumin as the standard. phoresis. This paper describes molecular and catalytic properties of the enzyme. Measurementof molecular weight. Molecular weight was measured by gel filtration on a column (2.2 x 79.5cm) of Sephacryl S-200 by the method of Andrews.4' Elution was Materials and Methods done with 50mMpotassium phosphate buffer, pH 7.4, containing 0.5m NaCl. The low rate was adjusted to 10ml Materials. Sephacryl S-200 was obtained from cm^hr"1, and 3.0ml fractions were collected. Five pro- Pharmacia Fine Chemicals, Sweden. Phosphodiesterase tein standards were used: bovine liver catalase (mol. wt. was purchased from Sigma Chemical Co., U.S.A. Other 232,000), rabbit muscle aldolase (mol. wt. 158,000), bovine chemicals were of analytical grade available from com- serum albumin (mol. wt. 67,000), hen egg albumin (mol. mercial sources. wt. 45,000), and bovine pancreas chymotrypsinogen A (mol. wt. 25,000). Vo was measured with Blue Dextran Preparation of nucleoside oxidase. Purified nucleoside 2,000. The molecular weights of the subunits were esti- Abbreviations: MES, 2-(A^-morpholino) ethanesulfonic acid; SDS, sodium dodecyl sulfate. 1672 Y. Isono, T. Sudo and M. Hoshino mated by sodium dodecyl sulfate (SDS)-acrylamide gel 5C4-300 column (04.5 x 150mm, Nacalai Tesque Inc.). electrophoresis by the method of Weber and Osborn.5) Subunis were eluted by a gradient of acetonitrile from 0 to The enzyme was denatured by treatment with 1%SDS 72% in 0.05% trifluroacetate solution. solution containing 1%2-mercaptoethanol at 100°C, lO min. Standard proteins: horse heart cytochrome c (mol. AMPassay. AMPwas assayed by HPLCunder the wt. 12,400), bovine pancreas chymotrypsinogen A (mol. following conditions: column, Fine SIL C18 (Japan wt. 25,000), bovine erythrocyte carbonic anhydrase (mol. Spectroscopic Co., Ltd. 04.5 x250mm); solvent, 10mM wt. 29,000), hen egg albumin (mol. wt. 45,000), bovine KH2PO4-H3PO4 (pH 3.0)/methanol (18 : 1, by volume); plasma albumin (mol. wt. 67,000), rabbit muscle phospho- flow rate, 1.5ml/min and detection, UVat 250nm. The rylase B (mol. wt. 97,400), and E. coli /?-galactosidase chromatogramwas recorded on an intergrator, and AMP (mol. wt. 116,000). was quantified on the basis of peak area using calibration Isoelectricfocusing. Isoelectric focusing was done at 4°C for 48hr on an LKB column (110ml) containing 1% Results Carrier Ampholite of pH 3.5 x 10.0 (LKB-Produkter AB, Sweden) by the method of Vesterberg and Svensson.6' Molecular properties of nucleoside oxidase Spectrophotometric measurements. Spectrophotometric 1) Molecular weight and subunit structure. As measurements were done with a Shimadzu model UV-240 shown in Fig. 1, the molecular weight of the recording spectrophotometer. enzyme was 130,000 by Andrew's gel filtration method on Sephacryl S-200. The subunit struc- Measurementof metals. Total iron was measured using 1,10-phenanthroline and atomic absorption with a Hitachi ture was analyzed by SDS-gel electrophoresis. atomic absorption spectrophotometer, model 180-50. There were four bands of stained protein, Non-heme iron was extracted with trichloroacetic acid, designated tentatively as subunits a, /?, y, and and the liberated iron was measured as ferrous 1,10- 3. By calibration with several standard pro- phenanthroline complex, as described by Massey8) teins, their molecular weights were 76,000, (Method A). Non-heme iron was also extracted into 33,000, 18,000, and 14,000. ethanol after reduction with sodium dithionite and was afterwards measured with 4,7-diphenyl-1,10-phenan- The purified enzymeprotein was also ana- throline as described by Doeg and Ziegler9) (Method lyzed by reverse-phase HPLCusing a Cosmosil B). 5Q-300. Figure 2 shows typical elution pat- Other metal contents of nucleoside oxidase were mea- terns of the enzyme protein at pH 7.0, 3.4, and sured by atomic absorption. Analytical grade metal salts wereused as standards. Measurement of labile sulfide. Labile sulfide was esti- mated by the method of King and Morris10' with the following minor modifications. A sample of nucleoside oxidase was made up to a volume of0.7ml by the addition of water in a centrifuge tube with ajoint stopper. To this, 0.5 ml of alkaline zinc reagent (freshly prepared by adding 5 volumes of2.6% zinc acetate to 1 volume of6% NaOH) was added. After the tube was stoppered, and shaken for 1 min, 0.1 ml of 0.02m A^N'-dimethyl-p-phenylendiamine sulfatein 7.2n HC1and0.1 ml of0.03m FeCl3 in 1.2n HC1 were added to the tube in rapid succession. After 1 hr, 1.0ml of water was added, and the precipitate was re- moved by centrifugation. The color of the supernatant Fig. 1. Measurement of the Molecular Weight of solution was measured at 670nm against a reagent blank. Standard solutions of sodium sulfide were prepared as Nucleoside Oxidase by Filtration on Sephacryl S-200. described by King and Morris.10' The enzyme (Z) and protein standards (A, bovine liver catalase; B, rabbit muscle aldolase; C, bovine serum albumin; D, hen egg albumin; and E, bovine pancreas Preparations ofsubunits a,p,y, and 3. Subunits a, y, and p-S complex were prepared by reverse-phase HPLCusing chymotrypsinogen A) were chromatographed at 4 °C on a two chromatography pumps, Waters model 6000Aand column (2.2 x 79.5 cm) equilibrated with 50 mMphosphate Waters model 660, a solvent programmerwith a Cosmocil buffer, pH 7.5, containing 0.5m NaCl. Properties of Nucleoside Oxidase 1673 Fig. 3. Gel Permeation HPLCofDenatured Peak (a) on TSKgel G3000SW XL. Peak (a) was denatured by treatment with 1% SDS solution containing 0.05m dithiothreitol at 100°C, for lOmin, and put on a column of TSKgel G3000SWXL. Chromatographic conditions are as follows: eluent, 0.2m sodium phosphate buffer, pH 7.0, containing 0.2% SDS; flow rate, l.Oml/min; detection, absorbance at 280nm. Fig. 2. Reverse-phase HPLCof Nucleoside Oxidase on Cosmosil 5Q-300. Theenzymesolution wasput on a columnof Cosmosi] 5C4-300 (04.5 x 150mm). Elution (l.Oml/min) was done with a gradient ofacetonitrile from 0 to 72%in (1) 10mM phosphate buffer, pH 7.0; (2) 10mMphosphate buffer, pH 3.4; (3) 0.05% trifluoroacetate, pH 2.4. 2.4. At pH 7.0, the enzyme was eluted as a single peak, and this peak had the activity. Whenthe enzyme was eluted at pH 3,4, the peak that had the activity decreased with the appearance of three additional peaks, desig- nated (a), (b), and (c), and these three peaks had no activity. At pH 2.4 the peak of native Fig. 4. Absorption Spectra of Nucleoside Oxidase. enzyme protein disappeared and three peaks The protein concentration was 0.48mg per ml in 20mM remained. On these three chromatographies, MES buffer, pH 6.0, ( ), oxidized form; ( ), reduced form treated with sodium hydrosulfite; ( -.), the protein recoveries were almost 100%. The reduced form treated with inosine. The inset represents a relative ratio of peak area or peak ptotein was differential spectrum of the oxidized and inosine reduced 1.00 (a): 1.52 (b): 0.45 (c) or 1.00 (a): 1.66 (b): forms. 0.43 (c). These three peaks were electropho- resed on SDS gel. Peak (a) had two protein linked together by disulfide bonds as indicated bands identified as subunits f$ and (5, and peak by the following results.
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