Purification and some properties of NAD-degrading purine nucleosidase from Aspergillus niger MASAAKIKUWAHARA AND TOMOKO FUJII

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Department ofFood Science, Kagawu UniversiQ, Miki-cho, Kc~gatr7u761-07, Japan Received November 17, 1977 Kuwahara, M. & Hujii, T. (1978) Purification and some properties of NAB-degrading purine nucleosidase from Aq..ergi!leds niger. Can. J. Biochern. 56, 345-348 An enzyme which degrades NAB at the adenine-ribose linkage has been purified from the mycelial extract of Aspergillus niger. NADP, deamido-NAB, and purine nucleosides and nucleotides were also susceptible to the hydrolytic cleavage. Pyrimidine- and nicotinamide-ribose linkages were not attacked. The substrate specificity showed that the enzyme may be classified ;as a N-ribosyl-purine ribohydrolase (EC 3.2.2.1). The enzyme had a maximum activity in the pH range of 4.8-4.5 toward NAD. The K, values for NAD, 5'-AMP. and inosine were 3.0,2.9, and 1.6 mM respectively. Muwahara, M.& Fujii, T. (1978) Purification and some properties of NAD-degrading purine nucleosidase from Aspergillus niger. Can. J. Biuchem. 56,345-348 k s extraits de mycelium d'Aspergi4lus niger, nsus avons purifii une enzyme qui degrade le NAE) B la liaison adenine-ribose. LRs substances suivantes sont aussi susceptibles d e clivage hydrolytique : NADP, desamido-NAB, nucleosides et nucl6stides puriques. Les liaisons pyrimidine- et nicotinamide-ribose ne sont pas attaquees. La spicificiti vis-h-vis le substrat perme t d e classifier cette enzyme comme une N-ribosyl-purine ribohydrolase (EC 3.2.2.1). L'activitC d e I'enzyme B I'kgard du NAE) est maximum entre les pH 4.8 et 4.5. Les valeiars de K, pour le NAE), le 5'-AMP et I'inosine sont respectivement d e 3.0,2.9 et 1.6 mM. [Traduit par le journal]

acid ribonucleotide, and nicotinic acid ribonucleoside were prepared using the method of Honjo and Nishizukw (8). WicotinWAD contains two riboside linkages susceptible to amide ribonucleoside was prepared from nicotinamide riboenzymatic or chemical hydrolysis. The nicotinamide- nucleotide with intestinal alkaline phosphomonoesterase ribose linkage is subject to hydrolysis with NAD gly- (Bsehringer und Soehne) according to the method of Maplan and cohydrolases which are widely distributed in mam- Stolzenbach (9) with a slight modification. BEAE-cellulose was malian tissues (1) and which are also found in certain mi- obtained from Whatman, Sephadex G-200 and DEAE-Sephadex crsorg~nisms(2, 3, 4). The adenine-ribose linkage can A-50 were from Pharmacia. The apparatus and carrier ampholite be split chemically with dilute acid (5). An enzyme activ- for isoelectric focusing were products of LMB-Rodukter. Elecity which spHits the linkage to produce adenine and trofocr~singwas carried out according to the standard procedure (10). The linear sucrose gradient containing the enzyme and NAmRDPR was first demonstrated in the extract of carrier ampholite (pH 3-6) was prepared using the LMB gradient Aspergillus niger (AKU 3302) (6). Tracer experiments mixer. Focusing was carried out at 404)-450V for specific indicated that growing mycelia of the mold secreted periods. Upon elution of the column, each fraction (2 mi) was WAmRDBR into the medium when either nicotinic acid assayed for its enzyme activity. or nicotinamide was added as a precursor of NAD synAspergillus nilper (AMU 3302) was grown in a medium conthesis (7). These results suggest the presence of a mvel taining (per litre): glucose, 50g; peptone, 5g: yeast extract, 2g: KH2PQ,,2g;(NH,),S0,, 2g, and MgSB,.7H28,0.5g. T h e p H NAD-degrading reaction in the mold. In the present report, we describe the purification of a of the medium was 5.5. Twenty-two litres of the medium placed purine nucleosidase type enzyme which catalyzes the in a 3 0 1 jar fermentor was inoculated with I C of the subculture which had been grown in a flask at 28°C for 48 h with rotary hydrolysis of WAD at the adenine-ribose linkage and shaking. Growth was at 30°C for about 40 h under aeration. The some of its properties. harvested mycelia were washed with distilled water. The yield of mycelia was about 50g (wet weight) per litre of the medium. Materials and Methods The standard enzyme assay mixture contained 12.5pmol of P-NAD, a-NAP), P-NABP, and p-nicotinamide ribonu- NAD, 50 pmol of acetate buffer, pH 4.0, and enzyme in a final cleotide were obtained from Sigma. Deamido-NAD, nicotinic volume of 0.5 mi. Incubation was a t 37°C for 90min and the reaction was terminated by heating the mixture at 1W0C for ABBREVIATIBNS: NAD, nicotinamide adenine dinucleotide; I min. Enzyme activity was assayed by determining the amount NADP, nicotinamide adenine dinucleotide phosphate; of adenine formed from NAD as follows. A 0.05-ml diquot of the Deamido-NAB, nicotinic acid adenine dinucleotide; AMP, mixture was spotted on Toyo filter paper No. 53 and chromatoadenosine monophosphate; GMP. guanosine monophosphate; graphed using n-butanol saturated with 3% ammonia as the IMP, inosine monophosphate; XMP, xanthosine monophos- solvent. The spot of adenine was detected under ultraviolet light phate; UMP, u~idine monophosphate; CMP, cytidine and cut from the chromatogram. The adenine was extracted monophosphate; Tads, Tris(hydrc~xymethyl)aminomethane; with 5 ml of 0.01 hr HCI at 37°C for 3 h and the amount was NAmRDPW. nicotinamide ribose diphosphate ribose. calculated based on the absorbance at 260 nm using the E value of

Introduction

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CAN. J. BIOCHEM. VOL. 56, 1978

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TABLE 1. Purification of the purine nucleosidase

Step

Fraction

Total protein, mg

1 2

Crude extract Frotarnine treatment Ammonium sulfate treatment First DEAE-cellulose chromatography Sephadex G-2O(B chromatography Second DEAE-cellulose chromatography DEAE-Sephadex chromatography First isoelectric focusing Second isoelectric focusing Sephadex G-200 chromatography

26000 20840 1150 245 46 24 18 3.9 0.8 0.2

3 4 5 6 7 8 9 10

12.7 x lo3. One unit of enzyme was defined as the amount of enzyme which produces 1 pmol of adeninelminat 3TC. Specific activity was expressed as units per milligram of protein. Protein was determined by the method of Lowry e t a / .(1 I), with crystalline bovine serum albumin as the standard, or by A,,, rneasurements (steps 8, 9, and 18). To assay the enzyme with other substrates, free bases corresponding to the substrate nucleosides or nucleotides were separated and detern~inedby the assay procedure given above.

Results and Discussion Pirr.$cation q f the Enzyme All steps except step 3 were carried out at 0-5'6 and the buffers contained lo-' M 2rnercaptoethanol. Sfep 1 Mycelia were ground in a chilled mortar with quartz sand and 0.81M Tris-PIC1 buffer (pH 7.2). The thick suspension was centrifuged for 30min at 9680g and the supernatant fluid was retained. Step 2 One and five-tentlls rnillilitres of 1% protamine sulfate solution (pH 7.0) per 100 rng of the protein was added to the crude extract with stirring. After 38 min, the precipitate was removed by centrifugation. Stq3 The supernatant solution was brought to 95% saturation with ammonium sulfate. The pH was kept at about 7.8 by the addition of 3 N ammonia. The precipitate was removed by centrifugation. The supernatant solution was concentrated to one-fifteenth its original volume in a rotary evaporator below 33°C. The precipitate of arnrnonium sulfate was removed by filtration and the filtrate was dialyzed against (6.025M Tris-HC1 buffer (pH 7.2). Step 4 The enzyme solution was applied to a DEAE-cellulose column (3 x 66 cm) equilibrated with the dialysis buffer. After the column was washed thoroughly with the same buffer, the enzyme was eluted with a linear gradient from 0 to 0.4 M NaCI. The fractions with enzyme activity were pooled and concentrated about 35-fold in an Amicon ultrafiltration ceH equipped with a ultrafiitration membrane ( D i d o PM 30). The enzyme solution was dialyzed against 0.025M Tris-HC1 buffer (pH 7.2).

Total units

Specific activity

Yield,

2630 2090 1210 1 B 10 830 580 440 135 38 11

0.10 0.10 I .05 4.53 18.04 24.16 24.44 34.62 47.50 55.00

100 79.5 46.0 42.2 31 .Q 22.0 16.7 5.1 1.4 0.4

%

Step 5 The enzyme solution was applied to a column (2.5 x 68cm) of Sephadex G-280 equilibrated with 0.025M Tris-HCl buffer (pH 9.2). The enzyme fraction eluted with the same buffer was pooled and concentrated about 10-fold by ultrafiltration. Step 6 The enzyme was applied to a DEAE-cellulose column (1.8 x 4'7 cm) equilibrated with 0.025 M Tris-HCl buffer (pH 7.2) and chromatographed as described in step 4. The active fractions were combined and concentrated about 30-fold by ultrafiltration. Step 7 The enzyme solution was applied to a DEAESephadex A-50 column (1.5 x 40 cm) equilibrated with 0.025 A4 Tris-HC1 buffer (pH 7.2). After the column had been washed with the same buffer, the enzyme was eluted with the buffer containing 8.15 M NaCI. Fractions containing enzyme activity were pooled and concentrated about 18-fold by ultrafiltration. Step 8 The enzyme solution was applied to the isoelectric focusing column (118ml) by incorporating the enzyme into the dense solution of the sucrose gradient. Focusing was carried out for 48h at 450V. Active fractions (isoelectric peak at pH 4.1) were combined and dialyzed against 0.025 M Tris-HC1 buffer (pH 7.2). Step 9 The enzyme solution was again applied to the isoelectric focusing column as described in step $ and focusing was carried out for 90h at 400V. Active fractions (isoelectric point at pH 3.9) were pooled, concentrated about $-fold by ultrafiltration, and dialyzed against 0.025 M Tris-HC1 buffer (pH 7.2). Step 10 The enzyme was applied to a column (1.2 x 25 cm) of Sephadex G-208 equiiibrated with the dialysis buffer and was eluted with the same buffer. Fractions with enzyme activity were combined and concentrated about 15-fold by ultrafiltration. Table 1 summarizes the yieids and specific activities

347

KUWAHARA AND FUJI1

TABLE 2. Substrate specificity

during purification. Approximately 550-fold purification was achieved with a yield of 0.4%. Properties

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The purified enzyme was shown to be homogeneous by the criterion of disc gel electrophoresis (Fig. 1). It was an acidic protein with an isoelectric point of p H 3.9. The activity of the enzyme with various ribonucleosides and i-ibonucIeotides a s the substrate is shown in Table 2. An important characteristic of A . niger enzyme is that it degrades NAD derivatives selectively at the c?$enine-ribose linkage. Our previous report demonstrated that NAD was stoichiometrically degraded to adenine and nicotinamide ribose diphosphate ribose (6). The inertness of the nicotinamide ribonucleotide and nicotinamide ribonucleoside indicated the stability of the nicotinamide-ribose linkage of NAB toward the action of the enzyme. The a-isomer of NAD was much more actively degraded than was the /3-NAD; whereas, deamido-NAD and WADP were less actively degraded. The enzyme showed extremely high activity toward inosine and 5'-IMP in the substrates tested. The other purine ribonucleosides and ribonucleotides were less active substrates. En contrast, pyrimidine derivatives were not at tacked. The enzyme has a maximum reactivity in the pH range

Substrate NAB NAD (a-isomer) NABP Ileamido-NAD Nicatinamide ribsnuelestide Nicotinic acid ribonucleotide Nicstinamide ribonucleoside Nicotinic acid ribonueleoside Adenosine 5'-AMP 2'-AMP 3 '-AMP Cuanosim 5'-GMP lnosine 5'-IMP Xanthosine 5'-XMP Usidine

Relative activity, %

Base firmed

Adenine Adenine Adenine Adenine

Adenine Adenine Adenine Guanine Guanine Hypoxanthine Hypoxanthine

5 '-UMP

Cytidine 5'-CMP --

NOT~ The : reaction mixture contained 5 pmol of a substrate, 50 pmol o f acetate buffer (pH 4.0), and 0.033 U (0.6 pg o f protein) of the enzyme. Incubation was at 37'6 for 30 mill.

of 4.0-4.5 toward NAD. The K , value was calculated as 3.0mM for NAB. The p H optimum was around 3.5 with 5'-AMP and inosine. K , values for 5' -AMP and inosine were 2.9 mM and 1.6 mM respectively. The enzyme activity was stable over a wide pH range from 3.0 to 9.0 for 90 min of incubation a t 37°C. Ethylenediaminetetraacetic acid, sodium fluoride, sodium arsenate, sodium azide, a , a ' -dipyrid yl, ethylmaleimide, and iodoacetate, each a t a final concentration of IOmM, did not affect enzyme activity. However, IlgC1, a t a find concentration of 10 mM caused 19% inhibition; at 1 mM, the inhibition was less than 5%. Divalent cations such a s Mg2+, Mn2+, @ua+,and Ca2+had no effect on the activity. Nucleosidases have been partially purified from several fungi. The nucleosidase of A . oryzae (12) apparently differs from A . niger enzyme in that it is strictly specific for 6-hydroxypurine ribonucleosides and their 5'monophosphate. Substrate specificity of the A . niger enzyme indicates a similarity between the enzyme and the extracellular nucleosidase a from several species of Aspergillus and Penicilliunr (13); the latter enzyme having been shown to have a high specificity for purine ribonucleosides. However, no activity of A . niger enzyme toward xanthosine was detected in contrast with nucleosidase a for which xanthosine was the preferred substrate. Nucleosidase a has not been purified in a homogeneous state and its activity toward NAD and other pyridine nucleotides has not been tested. Asper-gilFIG. 1. Disc gel e%ectrophoresisof the purine nucleosidase. lus niger enzyme also differs from bacterial nucleoThe purified enzyme preparation ( I f pg of protein) was elec- sidases (14, 15) with respect to its substrate specificity and pH dependence of the activity. trophoresed at pH 9.3 and 3 mA for 90 miin.

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CAN. J. BlOCHEM. VOL. 56, 1978

In summary. A . niger enzyme is thought t o be included in the category of N-ribosyl-purine ribohydrolase and is unique in that it has activity toward N A D and its derivatives, It would he unwise at present t o assume a physiological significance for this enzyme in the metabolism of NAD. However, the presence of nicotinamide ribose diphosghate ribose, a product of N A B degradation, in the mycelium and culture filtrate of A. p ~ i g ~(7) r = indicates some involvement of the enzyme in the N A B metabolism pathway. Another possible significance of the enzyme is the degradation of NAD, a s wet1 a s other adenine derivatives, t o supply adenine moieties for the salvage synthesis of nucleic acids. The fate of the adenine derived from N A B is currently under investigation.

Acknowledgements We wish t o thank Professor K. Yamanaka of Kagawa University for his advice and valued discussion. Thanks are also due to S . Kui-oki and %. hlatsurnura for their assistance. 1. Zatrnan, L. J., Kaplan, N. Q . , Cslswick, S. P. & Ciotti, M. M. (1954) J. Biol. Chem. 209,453-446s 2. Sarrna, D. S. R., Raj~dakshrni. C. & Nason, A. 11964) Biochim. Biophys. Acta 81,3 11-322

3. Kaplan, N. O., Colowick, S. P. & Nason, A. (1951) J.Bio8. Chena. 191,473-4633 4. Stathakos, D., Isaakidsu, I. & Thornou, H. (1973)Biockim. Biophys. Acta 302,80-89 5. Peleiderer, G., Sann, E. & Brtanderl, F. (1963) Biochim. Biophys. Acttz 73,39-49 6. Kuwahara, M. & Tsukarnsto, M. (1975)Agric. Biob. Ct'zern. 39, 1975- 1980 7. Kuwahara, M. (1976) Agric. Biod. Chem. 40, 1573-1580 8. Honjo, T. & Niskizuka, Y. 11971) in Methods in Enzyrnobo g ~vol. , 18B, pp. 132-137, Academic Press, New York 9. Kaplan, N. 8. & Stolzenbach, G. E (1957) in Methods in Dazyrreology, vol. 3, pp. 899-985. Academic Press, New York 18. Vesterberg, 8. (1971) in Methods in Enzymology, vol. 22, pp. 389-412, Academic Press, New York B 1. Lowry, 8.W., Rosebrough, N. J., Farr, A. L. & Randail, R. J. (1951) J . Biol. Chem. 193,265-275 12. Kuninaka. A. 41956) Bull. Agt-ic. Chenz. Soc. Jpn. 23, 281-288 13. Weese, E. T. (1968) Cun. J . Microbial. 14,377-382 14. Takagi, Y. & Hsrecker, B. L. (1957) J. Biol. Chern. 225, 77- 86 15. Terada, M., Tatibana, M. & Hayaishi. 8. 619671 J , B i d . Chem. 242,5578-5585