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A new method for the assay of poly(adenosine diphosphate ribose) glycohydrolase activity
ANALYTICAL
BIOCHEMISTRY
A New
Method
diphosphate
66,
434-445
(1975)
for the Assay ribose)
of Poly(adenosine
Glycohydrolase
Activity
LUIS 0. BURZIO, PATRICIO T. RIQUELME, AND S. S. KOIDE Institute of Biochemistry, Faculty of Sciences, Austral University, Valdivia, Chile, and The Population Council, The Rockefeller University New York, New York 10021
Received October 3 1, 1974; accepted January 24, 1975 Poly(adenosine diphosphate ribulose) [poly(ADP-Rib)] glycohydrolase activity was determined by measuring the amount of ADP-Rib hydrolyzed from polymers of ADP-Rib as substrate. In principle, the method consists of incubating oligomers or polymers of [W]ADP-Rib with testis glycohydrolase. The reaction was stopped by the addition of a suspension of Dowex 1X-2 formate in H,O (1:3,v/v) which adsorbed monomers and oligomers of ADP-Rib. The adsorbed [W]ADP-Rib was selectively extracted from the resin with 6 M formic acid. The amount of [W]ADP-Rib was estimated by measuring the radioactivity in aliquots of formic acid extract. Oligomers or polymers of ADP-Rib can be utilized as substrates since the reaction rates were the same with either compound. A method to determine phosphodiesterase and glycohydrolase activities was established. These two enzymic activities were distinguished by treating the products of the reactions with alkaline phosphatase and by differential extraction of the adsorbed reaction products on Dowex with 0.5 M and 6 M formic acid.
Eukaryotic cells contain a nuclear enzyme that catalyzes the conversion of NAD into poly(ADP-Rib)’ with the concomitant elimination of nicotinamide (I-5). The repeating units of ADP-Rib are linked together by a glycosidic bond of ribosyl-1,2-ribose (3,5). The characteristics of the polymer and properties of the polymerizing enzyme have been reported (6-9). This biopolymer is hydrolyzed by two types of enzymes, namely, phosphodiesterase and glycohydrolase. The pyrophosphate bond is hydrolyzed by venom and rat-liver nuclear phosphodiesterase (2,4,10,11). The products of the reaction are AMP, PR-AMP and ribose-5’-phosphate. The chain length of the polymer can be calculated from the relative amounts of PR-AMP and AMP (12). Recently, a new enzymatic reaction has been reported which splits the ribose-ribose linkage between two ADP-Rib units with the release of only ADP-Rib 1 Abbreviations used: ADP-Rib, adenosine diphosphate ribose; Ad, adenosine; AMP, adenosine monophosphate; PR-AMP, phosphoribosyl adenosine monophosphate or 2’-(5’ phosphoribosyl)-5 ’ AMP;R-Ad, 2’-(ribosyl)-adenosine; TCA, trichloroacetic acid. 434 Copyright All rights
@ 1975 by Academic Press, Inc. of reproduction in any form reserved.
ASSAY
OF
POLY(ADP-RIB)
GLYCOHYDROLASE
435
(13-l 6). This enzyme was designated as poly(ADP-Rib) glycohydrolase (13-16). Glycohydrolase activity had been determined by measuring the hydrolysis of radioactive poly(ADP-Rib) (13-16). The rate of the reaction can be followed by measuring the amount of TCA-precipitable polymers remaining after incubation with the enzyme. This method has been used to assay for phosphodiesterase and glycohydrolase activities (1 1,13,14,16). In this communication, an assay method to measure glycohydrolase and phosphodiesterase activities was worked out by determining the amount of ADP-Rib formed. Polymers and oligomers of [llC]ADP-Rib can be used as substrate. By the proposed method, phosphodiesterase and glycohydrolase activities can be distinguished. MATERIALS
AND
METHODS
[ AdenosineJ4C] NAD+ (159 mCi/mole) was purchased from AmershamlSearle. Adenosine, AMP, ADP-Rib and NAD+ (Grade V) were obtained from Sigma Chemical Co. Dowex 1X-2 (200-400 mesh, chloride form) was converted to the formate form according to standard procedure and resuspended in distilled water. Venom phosphodiesterase from Croralus adamanteus, Escherichia coli phosphatase, micrococcal nuclease, deoxyribonuclease I (DNase I) and pancreatic ribonuclease (RNase) were purchased from Worthington Biochemicals Corp. Other reagents were of analytical grade. Polymers and oligomers of ADP-Rib were prepared by using [ adenosineJK]N AD+ according to the following procedure. Rat liver nuclei were prepared as described in a previous report (17). The nuclei preparation (5 mg of protein/ml) was incubated in a medium containing 80 mu Tris-HCl (pH S.O), 0.5 mM EDTA, 10 mM MgCl,. and 0.5 mM [14C]NAD+ (1 pCi/pmole). The mixture was incubated at 25°C for 30 min. The reaction was stopped by the addition of cold TCA to a final concentration of 5%. The precipitate was collected by centrifugation. The sediment was washed once with 5% TCA and three times with 95% ethanol. After the final wash, the pellet was resuspended completely in 0.1 N NaOH and incubated for 60 min at 3’7°C. To the mixture Tris was added to a final concentration of 50 mM. The mixture was neutralized with concentrated HCI. Pronase was added to this suspension to a final concentration of 50 pg/ml and incubated for 4.5 min at 37°C. At the end of this initial incubation the same amount of Pronase was added, and the mixture was incubated for an additional 45 min. The solution was centrifuged at 15 ,OOOg for 15 min to remove any insoluble material formed. The supernatant fluid was deproteinized by extraction with a mixture of chloroform:isoamyl alcohol (24: 1, v/v) three times (18). To
436
BURZIO,
RIQUELME
AND
KOIDE
the aqueous layer, sodium acetate was added to a final concentration of 0.2 M (PI-I 5.0). Absolute ethanol (2.5 volumes) was added to precipitate the polymers and oligomers. The mixture was left standing for 24 hr at -20°C. The precipitate was collected by centrifugation. The sediment was washed twice with 70% ethanol and dried under vacuum. The dried material was dissolved in 50 mu Tris-HCI (pH 7.5). To the mixture, MgCl, and CaCI, solutions were added to a final concentration of 10 mr~,~ and I mu, respectively. To eliminate contaminating nucleic acids, DNase I and pancreatic RNase were added to a final concentration of 100 pg/ml. The mixture was incubated for 1 hr at 37°C. To the mixture, micrococcal nuclease was added to a final concentration of 100-200 units/ml. The mixture was incubated for 1 hr at 37°C. Pronase was added to a final concentration of SO pg/mI and the mixture incubated for 1 hr at 37°C. Upon completion of incubation the solution was deproteinized as described above. The polymers and oligomers were precipitated by the addition of 2.5 volumes of absolute ethanol at pH 5. The precipitate was collected by centrifugation. The sediment was dissolved in 25 mu Tris-HCl (pH 7.3), 1 M NaCl. The solution was applied to a column of Sephadex G-50 (90 X 1.5 cm). The column was equilibrated and eluted with the same buffer. A representative pattern of the gel filtration is shown in Fig. 1. A large part of the radioactive material was eluted in the void volume of the column. This material is precipitable with 10% TCA. The fractions (A) containing the radioactivity were pooled, and the polymer was precipitated with 2.5 volumes of ethanol. The remaining radioactive materials were composed of oligomers of ADP-Rib of varying chain-lengths. They were collected in two fractions (B and C), pooled and precipitated with ethanol as described above. The precipitate was collected by centrifugation at 15,000g for 15 min. Sediment from each fraction was dissolved in 10 mM potassium phosphate buffer (pH 7.0) to give a final radioactivity of about 300,000 cpmlml. In the present paper the fraction precipitated with 10% TCA was designated as poly(ADP-Rib) (Fraction A) and the remaining fraction as oligo(ADPRib) (Fractions B and C). The average chain-length of each fraction was determined with venom phosphodiesterase according to the procedure of Shima et al. (12). In the present study two polymers were used with the following characteristics: Chain length 26.5 and 35.2, respectively, and with the same specific activity of 1,780 cpm/nmole of ADP-Rib. Oligo(ADP-Rib) of the following chain-lengths were used: 12.3 and 4.9 (sp act, 1,780 cpmjnmole of ADP-Rib), and 9.7 units of ADP-Rib (sp act 3,350 cpmlnmole of ADP-Rib). The ratio of Aasonm to Azsonm of these polymers ranged between 0.25 and 0.29 which are in agreement with previous reports (19,20).
ASSAY
OF
POLY(ADP-RIB)
A nn-
20
GLYCOHYDROLASE
B
C
40
Fraction
437
60
number
FIG. I. Pattern of gel filtration of poly- and oligo([W] ADP-Rib) through Sephadex G-50. The polymers were digested with DNase, RNase and micrococcal nuclease to remove DNA and RNA contaminants and applied to a Sephadex G-50 column (I 3 X 90 mm) equilibrated with a medium containing 25 mM Tris-HCI (pH 7.5). 1 M NaCI. Fractions of 5 mutube with a Row rate of about 10 ml/hr were collected. Radioactivity in aliquots of 25 ~1 were determined. Total radioactivity, (O-O); TCA-precipitable radioactivity. (----).
ADP-Rib was separated by descending paper chromatography on Whatman No. 1 paper in a solvent system composed of isobutyric acid:concentrated NH,OH: water (66: 1:33, by volume) (12). Chromatography on Dowex 1X-2, formate form, was performed according to the method of Ueda er al. (14). [‘“Cl PR-AMP was prepared from oligo( [ 14C] ADP-Rib) by hydrolysis with venom phosphodiesterase ( 12). [‘“C] R-Ad was prepared by treating [‘“C] PR-AMP with E. coli alkaline phosphatase (12). The poly(ADP-Rib) glycohydrolase used in the present study was purified from rat testis. The procedure used in the purification of the enzyme and a description of its properties will be presented in another publication. The assay system for the determination of poly(ADP-Rib) glycohydrolase activity by the present method contained, in a total volume of 0.2 ml, 10 pmoles of potassium phosphate buffer (pH 7.0). 1 Fmole of 2-mercaptoethanol, oligo( (%] ADP-Rib), 3,500- 12,400 cpm. The reaction-mixture system was incubated for 10 min at 37°C. The reaction was stopped by the addition of 1 ml of Dowex suspended in H,O (1:3, v/v, freshly washed with H,O). The tubes were kept in ice. To each tube 1.2 ml of 12 M formic acid was added and the tubes shaken.
438
BWRZIO,
RIQUELME
AND
KOIDE
After standing for 5 min in ice with occasional shaking, the tubes were centrifuged at SOOg for 2 min. To 1.5 ml of the supernatant fluid, 10 ml of Tritol (66% of standard toluene cocktail for liquid scintillation and 33% of Triton X-100) were added. Radioactivity was measured in a Packard Scintillation Spectrometer. It was established that high concentrations of formic acid did not alter the counting efficiency for 14C. The total aqueous volume, after stopping the reaction with 12 M formic acid, was found to be 2.1 ml, which was estimated by using C3H]H20. This value was used in calculating the amount of [14C]ADP-Rib released. Protein was determined by the method of Lowry et al. (21). RESULTS
AND
DISCUSSION
The reaction mixture described under Materials and Methods was scaled up five times and, the total radioactivity of oligo([14C]ADP-Rib) used as substrate was 26,500 cpm. The mixture was incubated with 128 pg of enzyme for 15 min at 37°C. The reaction was stopped by diluting with 10 ml of cold HzO, and Ad, AMP, NAD+, ADP-Rib and ATP were added as markers. The entire mixture was loaded on a Dowex 1X-2, formate, column. The column was eluted as previously reported (13). The pattern of elution is depicted in Fig. 2. In the gradient region between water and 0.5 M formic acid (A-B). practically no radioactivity was eluted. In this region, Ad, NAD+ and AMP were eluted in that order. Between 0.5 and 5 M formic acid (B-C), about 45% of the radioactivity was eluted which corresponded to ADP-Rib (Fig. 2). When the column was eluted stepwise with 6 M formic acid (C) and 6 M formic acid plus 0.5 M ammonium formate (D) the remaining radioactivity (about 35%) was eluted. The latter radioactivity peak, eluted in the vicinity of ATP used as marker, corresponded to oligomers of ADP-Rib (13). The overall recovery of materials from the columns was about 80%. The present results indicate that ADP-Rib can be separated from oligo(ADP-Rib) with chain lengths shorter than the initial substrate by chromatography on Dowex 1X-2, formate form. A method for the determination of glycohydrolase activity by batchwise extraction of ADP-Rib from the Dowex resin was worked out. ADP-Rib and related nucleotides were adsorbed on Dowex resin and eluted with varying concentrations of formic acid (Fig. 3). Radioactive or nonradioactive ADP-Rib or other nucleotides were dissolved in the reaction mixture as described under Methods. To the mixtures 1 ml of the Dowex suspensions was added. The tubes were placed in ice for about 2 min and 1.2 ml of formic acid were added. The mixtures were centrifuged and the amount of radioactivity and the absorbance at 260 nm in the supernatant fluid were measured. About 70% of the Ad or the R-Ad (data not shown) was ex-
ASSAY
OF
POLY(ADP-RIB)
GLYCOHYDROLASE
ADP-Rib
0
20
40
80
60
100
JO
Fraction number FIG. 2. Chromatography of the reaction products from oligo([‘K]ADP-Rib) treated with poly(ADP-Rib) glycohydrolase on Dowex 1X-2, formate. column. Glycohydrolaae (128 pg) was incubated with 26.500 cpm of oligo([%]ADP-Rib) (IS nmoles of ADP-Rib) dissolved in 1 ml of 50 mu potassium phosphate buffer (pH 7.0). 10 mM ‘L-mercaptoethanol for 10 min at 37°C. Ad, AMP, NAD, ADP-Rib and ATP were added as markers and the entire mixture loaded on a Dowex column (1 x 32 cm). The column was eluted with a series of convex gradients between H,O and 0.5 M formic acid (A-B): between 0.5 and 6 M formic acid (B-C): with 6 M formic acid (C-D) and with 6 M formic acid and 0.5 M ammonium formate (0 to end). Fractions of 7 ml/tube were collected.
0
1
2
3
4 Formic
5 acid
6
7
8
(Ml
3. Stepwise elution of various nucleotides from Dowex, formate. with varying concentrations of formic acid. To the reaction mixtures (see Materials and Methods) containing equivalent amounts of Ad (a-&, AMP (O--O), or ADP-Rib (e-e), 1 ml of a Dowex. formate, suspension in H,O (1:3, v/v) was added. To the mixtures, 1.2 ml of formic acid were added to a final concentration as depicted. The amount of nucleotide extracted was determined in the formic acid extract. Values are given in percent. FIG.
440
BURZIO,
RIQUELME
AND
KOIDE
tracted with water and completely extracted with 0.5 M formic acid. AMP was extracted completely with 1 M formic acid while 85% of ADP-Rib and PR-AMP (data not shown) were extracted completely with 1 M formic acid. The amount of ADP-Rib recovered was independent of its initial concentration. When 0.05-2 pmoles of ADP-Rib were present in the incubation mixture, 80-85% of the nucleotides were extracted. The degree of extractability of ADP-Rib was found to be dependent upon the amount of Dowex resin used. A constant amount of ADP-Rib (0.2 pmole) in the reaction medium was mixed with 1 ml of Dowex in various proportions. The mixture was extracted with 6 M formic acid. At ratios of Dowex resins to H,O of 1: 1, 1:2, 1:3 and I :5 the amounts of ADP-Rib extracted were 68, 76, 84 and 83%, respectively. In subsequent experiments, a suspension containing one part of Dowex to three parts of H,O was utilized. It was necessary to establish that oligo(ADP-Rib) remained adsorbed to the Dowex resins at the concentration of formic acid used to extract ADP-Rib. To reaction mixtures containing varying amounts of oligo( [‘“Cl ADP-Rib), Dowex suspension was added and extracted with 6 M formic acid. With mixtures containing 3,540 cpm/2 nmoles, 8,850 cpm/5 nmoles, and 17,700 cpm/lO nmoles of oligo([14C]ADP-Rib) the amounts of radioactivity in the 6 M formic acid extracts were 110, 154 and 172 cpm, respectively. These values represent less than 3% of the amount of oligo( [‘“Cl ADP-Rib) used. The present established method was utilized to measure poly(ADPRib) glycohydrolase activity (Figs. 4A,B). The reaction rate as measured by the formation of ADP-Rib was linear up to 15 or 30 min of incubation depending upon the concentrations of oligo(ADP-Rib) used. At a substrate concentration of 15 FM (expressed as ADP-Rib), the reaction rate was linear for 15 min (Fig. 4A). At a higher concentration (35 PM) the reaction was linear up to 30 min. With poly(ADP-Rib) as substrate at a concentration of 17 ,UM the reaction was linear up to 30 min of incubation as shown in Fig. 4A (curve 3). It was observed that the rate of hydrolysis of oligo(ADP-Rib) was directly proportional to the amount of enzyme in the assay system (Fig. 4B). When the enzyme was omitted, the amount of radioactivity in the formic acid extract of the control system was 100 cpm based on ten separate experiments. This control value was subtracted from the values obtained with glycohydrolase in the assay system. Another control system regularly employed was a reaction mixture to which a known amount of [14C]ADP-Rib (usually about 2,000 cpm) was added and extracted with 6 M formic acid. The amount of radioactivity extracted represents the exact percentage of extraction for each experiment. This value was used to calculate the amount of ADP-Rib produced due to glycohydrolase activity.
ASSAY OF POLY(ADP-RIB)
441
GLYCOHYDROLASE
(fl~‘~/’ IO
20
30
IO
20
30
Time (min) Protern (rg) FIG. 4. (A) Time course of hydrolysis of poly- and oligo(ADP-Rib) by testis poly(ADPRib) glycohydrolase. Reaction rates were determined with oligo( [“Cl ADP-Rib) as substrates: 11,800 cpm, 6.9 nmoles of ADP-Rib (curve 1): 5.100 cpm, 3.1 nmoles of ADPRib (curve 2); with poly(ADP-Rib), 5,800 cpm, 3.4 nmoles of ADP-Rib (curve 3). Each system contained 14.5 ~g of glycohydrolase proteins. (6) Relationship of rate of [‘*C]ADP-Rib formed to concentration of poly(ADP-Rib) glycohydrolase. The substrate was oligo([‘“C]ADP-Rib) (6,100 cpm, 3.6 nmole of ADP-Rib). The experimental conditions are described under Materials and Methods.
ADP-rib AMP -
0
20
IO
Strip
30
number
Fto. 5. Paper chromatography of the radioactive products formed on hydrolysis of oligo([“C] GDP-Rib) with glycohydrolase. The reaction mixtures containing oligo( [“‘C]ADP-Rib) (6,100 cpm) and 29 m of glycohydrolase protein were incubated for 7 min (hatched area) and 20 min (clear area). The reaction was stopped with the addition of Dowex resin and the suspension extracted with 6 M formic acid. The formic acid extract was spotted on a Whatman No. I paper (50 X 2 cm) (500 ~1 of the 7-min extract and 780 ,uI of the 20 min-extract from a total of 2,100 ~1). and developed in a solvent system described under Materials and Methods for 15 hr. After drying, the paper was cut into l-cm strips and the radioactivity measured. ADP-Rib and AMP were included as markers. The solvent front of 7- and 20-min extracts is designated by arrows I and 2, respectively.
442
BURZIO,
RIQUELME
AND
KOIDE
. The radioactive derivatives in the 6 M formic acid extract were identified by paper chromatography (Fig. 5). Oligo( [ 14C] ADP-Rib) (I 2,200 cpm corresponding to 6.9 nmole of ADP-Rib) was incubated with 42 pug of glycohydrolase for 7 and 20 min. The reaction was stopped by the addition of Dowex resins and the mixture extracted with 6 M formic acid. About 150 pg of nonradioactive ADP-Rib was added as carrier. The formic acid extract was carefully applied to a sheet of Whatman No. I chromatography paper (50 X 2 cm). The paper was developed overnight in a solvent system as described under Materials and Methods. The chromatogram was dried, cut into 1-cm strips and each strip counted in a standard toluene-based scintillation cocktail. It was found that at 7 and 20 min of incubation the recoveries of the radioactivity with the ADPRib carrier applied to the paper were 87 and 92%, respectively. The identity of radioactive ADP-Rib was also established by using other chromatographic solvent systems (35). The action of venom phosphodiesterase on poly- or oligo(ADP-Rib) yielded two products, namely, AMP and PR-AMP, which can be dephosphorylated completely by E. co/i alkaline phosphatase (19). These dephosphorylated compounds (Ad and R-Ad) are poorly bound to Dowex and can be easily extracted with 0.5 M formic acid (see Fig. 3). On the other hand, ADP-Rib, the product of the glycohydrolase action, cannot be dephosphorylated by alkaline phosphatase and was not extracted from Dowex with 0.5 M formic acid (Fig. 3). These findings were utilized in the present method to distinguish between glycohydrolase and phosphodiesterase activity with oligo(ADP-Rib) as substrate (Table 1). After incubating 25 ,ug of glycohydrolase (with or without E. coli alkaline phosphatase) in the standard assay mixture for 10 min at 37°C the reaction was stopped by the addition of Dowex resins. The mixture was extracted with 0.5 M formic acid. The mixture was centrifuged, the supernatant fluid collected, and the sediment of Dowex extracted with 6 M formic acid. The amount of radioactivity in aliquots of 0.5 M and 6 M formic acid extracts was determined. When treated with glycohydrolase alone, the amount of radioactivity in the 0.5 M formic acid extract was barely detectable while that in the 6 M acid extract was high. This distribution in counts was not affected by prior treatment with E. coli alkaline phosphatase. When similar experiments were carried out with venom phosphodiesterase, about 20% of the radioactivity was extracted with 0.5 M formic acid and the remaining amount with 6 M formic acid. However, when alkaline phosphatase was included in the reaction mixture, most of the radioactivity was extracted with 0.5 M formic acid. Based on these results, the two reaction mixtures, one containing glycohydrolase plus phosphatase and the other containing phosphodiesterase plus phosphatase, were incubated as described above. At the end of the
ASSAY
OF
POLY(ADP-RIB) TABLE
443
GLYCOHYDROLASE I
DETERMINATION OF GLYCOHYDROLASE AND VENOM PHOSPHODIESTERASE ACTIVITIES Activitya (cpm) Enzyme systems __-~ A B C D E F
Control Glycohydrolase Glycohydrolase + alkaline phosphatase Venom phosphodiesterase Venom phosphodiesterase + alkaline phosphatase Combination of C + E
0.5 M” --~ --~ ---_I 88 132 104 428 1759 1642
6.0 M” 49 974 905 1060 141 876
n The composition of the reaction mixture was described in detail under Materials and Methods. The system contained 9,800 cpm of oligo(~C]ADPR) (3.1 nmoks as ADP-Rib) and 19 pg of glycohydrolase or 23 fig of venom phosphodiesterase. In experiments C. E and F. I .5 units of E. c&i alkaline phosphatase were added. The different enzyme systems were incubated for 15 min at 37°C and the reactions were stopped by adding the Dowex suspension. Each mixture was extracted when 0.5 M and 6.0 M formic as indicated in the text. The volume used for the determination of the amount of radioactivity was 0.8 ml. The values shown need to be multiplied by 2.62 to obtain the total amount of radioactivity present in 2.1 ml of each formic acid extract. Experiment A (control) was not incubated and represents the value obtained at time zero. Systems C and E were incubated separately and mixed and the reaction stopped with the Dowex suspension. b Final concentration of formic acid.
incubation period, both reaction mixtures were added to a tube containing 2 ml of Dowex resin suspension. The product of the reaction was extracted sequentiahy with 0.5 and 6 M formic acid as described above. The results as shown in Table 1 indicate that the radioactivity extracted with 0.5 M formic acid corresponded to phosphodiesterase activity (plus phosphatase), while the radioactivity extracted with 6 M formic acid reflected glycohydrolase activity. By the present method, glycohydrolase and phosphodiesterase activities can be distinguished by using a combination of alkaline phosphatase treatment and differential extraction of the reaction products with formic acid. Although the present method of assay is less sensitive than the previously reported procedure of TCA-precipitable poly(ADP-Rib) (13.16), the linearity of the reaction of the latter method was very brief (16), such that the activity of each batch of enzyme preparations had to be adjusted so that not more than 30-40% of the polymer used as substrate was hydrolyzed. In other words, the measured activities varied with the chain length of the polymer when assayed by the latter method. We observed that the activities differed widely when the same enzyme preparation was assayed with polymers containing 20.7 or 35.2 residues of ADP-Rib. These problems were eliminated in the present method. Very reproducible results were obtained when oligomers or polymers were
444
BURZIO,
RIQUELME
AND KOIDE
utilized. Moreover, since poly(ADP-Rib) glycohydrolases of calf thymus (16) and of testes (unpublished results) are exoglycohydrolytic enzymes, the present assay method of measuring the production of [‘“Cl ADP-Rib is a more direct determination of the enzymic activity than the TCAprecipitation method. The present method works only for exonucleolytic and not for endonucleolytic cleavage and is less expensive because oligomers can be used as substrates. The principle of the present method can be applied for determinations of other enzymic reactions, providing the substrate and the products of the reaction have significantly different affinities for the resin. In a preliminary study, exploratory experiments were carried out using Dowex 1X-2, chloride form. The method was not feasible because poly(ADPRib) and oligo(ADP-Rib) were poorly bound by the resin in the chloride form. The purification steps for polymers and oligomers of ADP-Rib as outlined in the present paper has the advantages of simplicity, rapidity and good yield, compared to the previously reported methods (12,19). The initial treatment with 0.1 N NaOH greatly improved the yield, and the purity of the final products was equivalent to that obtained by previous methods (12,19). ACKNOWLEDGMENTS The authors express their appreciation to Mrs. Gabriela E. Vizcarra for her excellent technical assistance. This work was partially supported by a grant of the Population Council (No. M73.71).
REFERENCES 1. Chambon, P., Weill, J. D., Doly, J., Strosser, M. T., and Mandei, P. (1966) Biochem. Biophys. Res. Commun. 25, 638443. 2. Nishizuka, Y., Ueda, K., Nakazawa, K., and Hayaishi, 0. (1967)J. Bid. Chem. 242, 3164-3171. 3. Reeder, R. H., Ueda, K., Honjo, T., Nishizuka, Y., and Hayaishi, 0. (1967) 1. Biol. Chem. 242,3 172-3 179. 4. Sugimura, T., Fujimura, S., Hasegawa, S., and Kawamura, Y. (1967) Biochim. Biophys. Acta 138, 438-441. 5. Fujimura, S., Hasegawa, S., Shimizu, Y., and Sugimura, T. (1967) Biochim. Biophys. Acta 145, 247-259. 6. Burzio, L., and Koide, S. S. (1972) FEBS Left. 20, 20-32. 7. Lehmann, A. R., and Shall, S. (1972) FEBS Left. 26, 181-184. 8. Roberts, J. H., Stark, P., and Smulson, M. (1973) Biochem. Biophys. Res. Commun. 52,43-50. 9. Janakidevi, K., and Koh, C. (1974) Biochemistry 13, 1327-1330. 10. Futai, M., and Mizuno, D. (1967)J. Biol. Chem. 242, 5301-5307. 11. Futai, M., Mizuno, D., and Sugimura, T. (1968) j. Biol. Chem. 243, 63254329. 12. Shima, T., Hasegawa, S., Fujimura, S., Matsubara, H., and Sugimura, T. (1969) J. Biol. Chem. 244, 6632-6635. 13. Miwa, M., and Sugimura, T., (1971)5. Eiol. Chem. 246, 6362-6364.
ASSAY OF POLY(ADP-RIB)
GLYCOHYDROLASE
445
14. Ueda, D., Oka, J., Narumiya, S., Miyakawa, N., and Hayaishi. 0. (1972) Biochem. Biophys. Res. Commun. 46, 5 16-523. 15. Miyakawa, N., Ueda, K., and Hayaishi, 0. (1972) Biochem. Biophys. Res. Commun. 49, 239-245.
16. Miwa, M., Tanaka, M., Matsushima, T.. and Sugimura, T. (1974) J. Biol. Chem. 249, 3475-3482. 17.
18. 19. 20. 21.
Burzio, L., and Koide, S. S. (1973) Biochem. Biophys. Res. Commtcn. 53, 572-579. Marmur, J. (1961) J. Mol. Biol. 3, 208-218. Sugimura, T., Yoshimura, N., Miwa, M., Nagai, H., and Nago. M. (197 I ) Arch. Biochem. Biophys. 147, 660-665. Shima, T., Fujimura, S., Hasegawa, S., Shimizu, Y.. and Sugimura. T. (1970) J. Bid. Chem. 245, 1327-1330. Lowry. 0. H., Rosebrough, N. J., Farr, A. L.. and Randall. R. J. ( 1951) J. fG(tl. Cl~em. 193, 265-275.
BIOCHEMISTRY
A New
Method
diphosphate
66,
434-445
(1975)
for the Assay ribose)
of Poly(adenosine
Glycohydrolase
Activity
LUIS 0. BURZIO, PATRICIO T. RIQUELME, AND S. S. KOIDE Institute of Biochemistry, Faculty of Sciences, Austral University, Valdivia, Chile, and The Population Council, The Rockefeller University New York, New York 10021
Received October 3 1, 1974; accepted January 24, 1975 Poly(adenosine diphosphate ribulose) [poly(ADP-Rib)] glycohydrolase activity was determined by measuring the amount of ADP-Rib hydrolyzed from polymers of ADP-Rib as substrate. In principle, the method consists of incubating oligomers or polymers of [W]ADP-Rib with testis glycohydrolase. The reaction was stopped by the addition of a suspension of Dowex 1X-2 formate in H,O (1:3,v/v) which adsorbed monomers and oligomers of ADP-Rib. The adsorbed [W]ADP-Rib was selectively extracted from the resin with 6 M formic acid. The amount of [W]ADP-Rib was estimated by measuring the radioactivity in aliquots of formic acid extract. Oligomers or polymers of ADP-Rib can be utilized as substrates since the reaction rates were the same with either compound. A method to determine phosphodiesterase and glycohydrolase activities was established. These two enzymic activities were distinguished by treating the products of the reactions with alkaline phosphatase and by differential extraction of the adsorbed reaction products on Dowex with 0.5 M and 6 M formic acid.
Eukaryotic cells contain a nuclear enzyme that catalyzes the conversion of NAD into poly(ADP-Rib)’ with the concomitant elimination of nicotinamide (I-5). The repeating units of ADP-Rib are linked together by a glycosidic bond of ribosyl-1,2-ribose (3,5). The characteristics of the polymer and properties of the polymerizing enzyme have been reported (6-9). This biopolymer is hydrolyzed by two types of enzymes, namely, phosphodiesterase and glycohydrolase. The pyrophosphate bond is hydrolyzed by venom and rat-liver nuclear phosphodiesterase (2,4,10,11). The products of the reaction are AMP, PR-AMP and ribose-5’-phosphate. The chain length of the polymer can be calculated from the relative amounts of PR-AMP and AMP (12). Recently, a new enzymatic reaction has been reported which splits the ribose-ribose linkage between two ADP-Rib units with the release of only ADP-Rib 1 Abbreviations used: ADP-Rib, adenosine diphosphate ribose; Ad, adenosine; AMP, adenosine monophosphate; PR-AMP, phosphoribosyl adenosine monophosphate or 2’-(5’ phosphoribosyl)-5 ’ AMP;R-Ad, 2’-(ribosyl)-adenosine; TCA, trichloroacetic acid. 434 Copyright All rights
@ 1975 by Academic Press, Inc. of reproduction in any form reserved.
ASSAY
OF
POLY(ADP-RIB)
GLYCOHYDROLASE
435
(13-l 6). This enzyme was designated as poly(ADP-Rib) glycohydrolase (13-16). Glycohydrolase activity had been determined by measuring the hydrolysis of radioactive poly(ADP-Rib) (13-16). The rate of the reaction can be followed by measuring the amount of TCA-precipitable polymers remaining after incubation with the enzyme. This method has been used to assay for phosphodiesterase and glycohydrolase activities (1 1,13,14,16). In this communication, an assay method to measure glycohydrolase and phosphodiesterase activities was worked out by determining the amount of ADP-Rib formed. Polymers and oligomers of [llC]ADP-Rib can be used as substrate. By the proposed method, phosphodiesterase and glycohydrolase activities can be distinguished. MATERIALS
AND
METHODS
[ AdenosineJ4C] NAD+ (159 mCi/mole) was purchased from AmershamlSearle. Adenosine, AMP, ADP-Rib and NAD+ (Grade V) were obtained from Sigma Chemical Co. Dowex 1X-2 (200-400 mesh, chloride form) was converted to the formate form according to standard procedure and resuspended in distilled water. Venom phosphodiesterase from Croralus adamanteus, Escherichia coli phosphatase, micrococcal nuclease, deoxyribonuclease I (DNase I) and pancreatic ribonuclease (RNase) were purchased from Worthington Biochemicals Corp. Other reagents were of analytical grade. Polymers and oligomers of ADP-Rib were prepared by using [ adenosineJK]N AD+ according to the following procedure. Rat liver nuclei were prepared as described in a previous report (17). The nuclei preparation (5 mg of protein/ml) was incubated in a medium containing 80 mu Tris-HCl (pH S.O), 0.5 mM EDTA, 10 mM MgCl,. and 0.5 mM [14C]NAD+ (1 pCi/pmole). The mixture was incubated at 25°C for 30 min. The reaction was stopped by the addition of cold TCA to a final concentration of 5%. The precipitate was collected by centrifugation. The sediment was washed once with 5% TCA and three times with 95% ethanol. After the final wash, the pellet was resuspended completely in 0.1 N NaOH and incubated for 60 min at 3’7°C. To the mixture Tris was added to a final concentration of 50 mM. The mixture was neutralized with concentrated HCI. Pronase was added to this suspension to a final concentration of 50 pg/ml and incubated for 4.5 min at 37°C. At the end of this initial incubation the same amount of Pronase was added, and the mixture was incubated for an additional 45 min. The solution was centrifuged at 15 ,OOOg for 15 min to remove any insoluble material formed. The supernatant fluid was deproteinized by extraction with a mixture of chloroform:isoamyl alcohol (24: 1, v/v) three times (18). To
436
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the aqueous layer, sodium acetate was added to a final concentration of 0.2 M (PI-I 5.0). Absolute ethanol (2.5 volumes) was added to precipitate the polymers and oligomers. The mixture was left standing for 24 hr at -20°C. The precipitate was collected by centrifugation. The sediment was washed twice with 70% ethanol and dried under vacuum. The dried material was dissolved in 50 mu Tris-HCI (pH 7.5). To the mixture, MgCl, and CaCI, solutions were added to a final concentration of 10 mr~,~ and I mu, respectively. To eliminate contaminating nucleic acids, DNase I and pancreatic RNase were added to a final concentration of 100 pg/ml. The mixture was incubated for 1 hr at 37°C. To the mixture, micrococcal nuclease was added to a final concentration of 100-200 units/ml. The mixture was incubated for 1 hr at 37°C. Pronase was added to a final concentration of SO pg/mI and the mixture incubated for 1 hr at 37°C. Upon completion of incubation the solution was deproteinized as described above. The polymers and oligomers were precipitated by the addition of 2.5 volumes of absolute ethanol at pH 5. The precipitate was collected by centrifugation. The sediment was dissolved in 25 mu Tris-HCl (pH 7.3), 1 M NaCl. The solution was applied to a column of Sephadex G-50 (90 X 1.5 cm). The column was equilibrated and eluted with the same buffer. A representative pattern of the gel filtration is shown in Fig. 1. A large part of the radioactive material was eluted in the void volume of the column. This material is precipitable with 10% TCA. The fractions (A) containing the radioactivity were pooled, and the polymer was precipitated with 2.5 volumes of ethanol. The remaining radioactive materials were composed of oligomers of ADP-Rib of varying chain-lengths. They were collected in two fractions (B and C), pooled and precipitated with ethanol as described above. The precipitate was collected by centrifugation at 15,000g for 15 min. Sediment from each fraction was dissolved in 10 mM potassium phosphate buffer (pH 7.0) to give a final radioactivity of about 300,000 cpmlml. In the present paper the fraction precipitated with 10% TCA was designated as poly(ADP-Rib) (Fraction A) and the remaining fraction as oligo(ADPRib) (Fractions B and C). The average chain-length of each fraction was determined with venom phosphodiesterase according to the procedure of Shima et al. (12). In the present study two polymers were used with the following characteristics: Chain length 26.5 and 35.2, respectively, and with the same specific activity of 1,780 cpm/nmole of ADP-Rib. Oligo(ADP-Rib) of the following chain-lengths were used: 12.3 and 4.9 (sp act, 1,780 cpmjnmole of ADP-Rib), and 9.7 units of ADP-Rib (sp act 3,350 cpmlnmole of ADP-Rib). The ratio of Aasonm to Azsonm of these polymers ranged between 0.25 and 0.29 which are in agreement with previous reports (19,20).
ASSAY
OF
POLY(ADP-RIB)
A nn-
20
GLYCOHYDROLASE
B
C
40
Fraction
437
60
number
FIG. I. Pattern of gel filtration of poly- and oligo([W] ADP-Rib) through Sephadex G-50. The polymers were digested with DNase, RNase and micrococcal nuclease to remove DNA and RNA contaminants and applied to a Sephadex G-50 column (I 3 X 90 mm) equilibrated with a medium containing 25 mM Tris-HCI (pH 7.5). 1 M NaCI. Fractions of 5 mutube with a Row rate of about 10 ml/hr were collected. Radioactivity in aliquots of 25 ~1 were determined. Total radioactivity, (O-O); TCA-precipitable radioactivity. (----).
ADP-Rib was separated by descending paper chromatography on Whatman No. 1 paper in a solvent system composed of isobutyric acid:concentrated NH,OH: water (66: 1:33, by volume) (12). Chromatography on Dowex 1X-2, formate form, was performed according to the method of Ueda er al. (14). [‘“Cl PR-AMP was prepared from oligo( [ 14C] ADP-Rib) by hydrolysis with venom phosphodiesterase ( 12). [‘“C] R-Ad was prepared by treating [‘“C] PR-AMP with E. coli alkaline phosphatase (12). The poly(ADP-Rib) glycohydrolase used in the present study was purified from rat testis. The procedure used in the purification of the enzyme and a description of its properties will be presented in another publication. The assay system for the determination of poly(ADP-Rib) glycohydrolase activity by the present method contained, in a total volume of 0.2 ml, 10 pmoles of potassium phosphate buffer (pH 7.0). 1 Fmole of 2-mercaptoethanol, oligo( (%] ADP-Rib), 3,500- 12,400 cpm. The reaction-mixture system was incubated for 10 min at 37°C. The reaction was stopped by the addition of 1 ml of Dowex suspended in H,O (1:3, v/v, freshly washed with H,O). The tubes were kept in ice. To each tube 1.2 ml of 12 M formic acid was added and the tubes shaken.
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After standing for 5 min in ice with occasional shaking, the tubes were centrifuged at SOOg for 2 min. To 1.5 ml of the supernatant fluid, 10 ml of Tritol (66% of standard toluene cocktail for liquid scintillation and 33% of Triton X-100) were added. Radioactivity was measured in a Packard Scintillation Spectrometer. It was established that high concentrations of formic acid did not alter the counting efficiency for 14C. The total aqueous volume, after stopping the reaction with 12 M formic acid, was found to be 2.1 ml, which was estimated by using C3H]H20. This value was used in calculating the amount of [14C]ADP-Rib released. Protein was determined by the method of Lowry et al. (21). RESULTS
AND
DISCUSSION
The reaction mixture described under Materials and Methods was scaled up five times and, the total radioactivity of oligo([14C]ADP-Rib) used as substrate was 26,500 cpm. The mixture was incubated with 128 pg of enzyme for 15 min at 37°C. The reaction was stopped by diluting with 10 ml of cold HzO, and Ad, AMP, NAD+, ADP-Rib and ATP were added as markers. The entire mixture was loaded on a Dowex 1X-2, formate, column. The column was eluted as previously reported (13). The pattern of elution is depicted in Fig. 2. In the gradient region between water and 0.5 M formic acid (A-B). practically no radioactivity was eluted. In this region, Ad, NAD+ and AMP were eluted in that order. Between 0.5 and 5 M formic acid (B-C), about 45% of the radioactivity was eluted which corresponded to ADP-Rib (Fig. 2). When the column was eluted stepwise with 6 M formic acid (C) and 6 M formic acid plus 0.5 M ammonium formate (D) the remaining radioactivity (about 35%) was eluted. The latter radioactivity peak, eluted in the vicinity of ATP used as marker, corresponded to oligomers of ADP-Rib (13). The overall recovery of materials from the columns was about 80%. The present results indicate that ADP-Rib can be separated from oligo(ADP-Rib) with chain lengths shorter than the initial substrate by chromatography on Dowex 1X-2, formate form. A method for the determination of glycohydrolase activity by batchwise extraction of ADP-Rib from the Dowex resin was worked out. ADP-Rib and related nucleotides were adsorbed on Dowex resin and eluted with varying concentrations of formic acid (Fig. 3). Radioactive or nonradioactive ADP-Rib or other nucleotides were dissolved in the reaction mixture as described under Methods. To the mixtures 1 ml of the Dowex suspensions was added. The tubes were placed in ice for about 2 min and 1.2 ml of formic acid were added. The mixtures were centrifuged and the amount of radioactivity and the absorbance at 260 nm in the supernatant fluid were measured. About 70% of the Ad or the R-Ad (data not shown) was ex-
ASSAY
OF
POLY(ADP-RIB)
GLYCOHYDROLASE
ADP-Rib
0
20
40
80
60
100
JO
Fraction number FIG. 2. Chromatography of the reaction products from oligo([‘K]ADP-Rib) treated with poly(ADP-Rib) glycohydrolase on Dowex 1X-2, formate. column. Glycohydrolaae (128 pg) was incubated with 26.500 cpm of oligo([%]ADP-Rib) (IS nmoles of ADP-Rib) dissolved in 1 ml of 50 mu potassium phosphate buffer (pH 7.0). 10 mM ‘L-mercaptoethanol for 10 min at 37°C. Ad, AMP, NAD, ADP-Rib and ATP were added as markers and the entire mixture loaded on a Dowex column (1 x 32 cm). The column was eluted with a series of convex gradients between H,O and 0.5 M formic acid (A-B): between 0.5 and 6 M formic acid (B-C): with 6 M formic acid (C-D) and with 6 M formic acid and 0.5 M ammonium formate (0 to end). Fractions of 7 ml/tube were collected.
0
1
2
3
4 Formic
5 acid
6
7
8
(Ml
3. Stepwise elution of various nucleotides from Dowex, formate. with varying concentrations of formic acid. To the reaction mixtures (see Materials and Methods) containing equivalent amounts of Ad (a-&, AMP (O--O), or ADP-Rib (e-e), 1 ml of a Dowex. formate, suspension in H,O (1:3, v/v) was added. To the mixtures, 1.2 ml of formic acid were added to a final concentration as depicted. The amount of nucleotide extracted was determined in the formic acid extract. Values are given in percent. FIG.
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tracted with water and completely extracted with 0.5 M formic acid. AMP was extracted completely with 1 M formic acid while 85% of ADP-Rib and PR-AMP (data not shown) were extracted completely with 1 M formic acid. The amount of ADP-Rib recovered was independent of its initial concentration. When 0.05-2 pmoles of ADP-Rib were present in the incubation mixture, 80-85% of the nucleotides were extracted. The degree of extractability of ADP-Rib was found to be dependent upon the amount of Dowex resin used. A constant amount of ADP-Rib (0.2 pmole) in the reaction medium was mixed with 1 ml of Dowex in various proportions. The mixture was extracted with 6 M formic acid. At ratios of Dowex resins to H,O of 1: 1, 1:2, 1:3 and I :5 the amounts of ADP-Rib extracted were 68, 76, 84 and 83%, respectively. In subsequent experiments, a suspension containing one part of Dowex to three parts of H,O was utilized. It was necessary to establish that oligo(ADP-Rib) remained adsorbed to the Dowex resins at the concentration of formic acid used to extract ADP-Rib. To reaction mixtures containing varying amounts of oligo( [‘“Cl ADP-Rib), Dowex suspension was added and extracted with 6 M formic acid. With mixtures containing 3,540 cpm/2 nmoles, 8,850 cpm/5 nmoles, and 17,700 cpm/lO nmoles of oligo([14C]ADP-Rib) the amounts of radioactivity in the 6 M formic acid extracts were 110, 154 and 172 cpm, respectively. These values represent less than 3% of the amount of oligo( [‘“Cl ADP-Rib) used. The present established method was utilized to measure poly(ADPRib) glycohydrolase activity (Figs. 4A,B). The reaction rate as measured by the formation of ADP-Rib was linear up to 15 or 30 min of incubation depending upon the concentrations of oligo(ADP-Rib) used. At a substrate concentration of 15 FM (expressed as ADP-Rib), the reaction rate was linear for 15 min (Fig. 4A). At a higher concentration (35 PM) the reaction was linear up to 30 min. With poly(ADP-Rib) as substrate at a concentration of 17 ,UM the reaction was linear up to 30 min of incubation as shown in Fig. 4A (curve 3). It was observed that the rate of hydrolysis of oligo(ADP-Rib) was directly proportional to the amount of enzyme in the assay system (Fig. 4B). When the enzyme was omitted, the amount of radioactivity in the formic acid extract of the control system was 100 cpm based on ten separate experiments. This control value was subtracted from the values obtained with glycohydrolase in the assay system. Another control system regularly employed was a reaction mixture to which a known amount of [14C]ADP-Rib (usually about 2,000 cpm) was added and extracted with 6 M formic acid. The amount of radioactivity extracted represents the exact percentage of extraction for each experiment. This value was used to calculate the amount of ADP-Rib produced due to glycohydrolase activity.
ASSAY OF POLY(ADP-RIB)
441
GLYCOHYDROLASE
(fl~‘~/’ IO
20
30
IO
20
30
Time (min) Protern (rg) FIG. 4. (A) Time course of hydrolysis of poly- and oligo(ADP-Rib) by testis poly(ADPRib) glycohydrolase. Reaction rates were determined with oligo( [“Cl ADP-Rib) as substrates: 11,800 cpm, 6.9 nmoles of ADP-Rib (curve 1): 5.100 cpm, 3.1 nmoles of ADPRib (curve 2); with poly(ADP-Rib), 5,800 cpm, 3.4 nmoles of ADP-Rib (curve 3). Each system contained 14.5 ~g of glycohydrolase proteins. (6) Relationship of rate of [‘*C]ADP-Rib formed to concentration of poly(ADP-Rib) glycohydrolase. The substrate was oligo([‘“C]ADP-Rib) (6,100 cpm, 3.6 nmole of ADP-Rib). The experimental conditions are described under Materials and Methods.
ADP-rib AMP -
0
20
IO
Strip
30
number
Fto. 5. Paper chromatography of the radioactive products formed on hydrolysis of oligo([“C] GDP-Rib) with glycohydrolase. The reaction mixtures containing oligo( [“‘C]ADP-Rib) (6,100 cpm) and 29 m of glycohydrolase protein were incubated for 7 min (hatched area) and 20 min (clear area). The reaction was stopped with the addition of Dowex resin and the suspension extracted with 6 M formic acid. The formic acid extract was spotted on a Whatman No. I paper (50 X 2 cm) (500 ~1 of the 7-min extract and 780 ,uI of the 20 min-extract from a total of 2,100 ~1). and developed in a solvent system described under Materials and Methods for 15 hr. After drying, the paper was cut into l-cm strips and the radioactivity measured. ADP-Rib and AMP were included as markers. The solvent front of 7- and 20-min extracts is designated by arrows I and 2, respectively.
442
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KOIDE
. The radioactive derivatives in the 6 M formic acid extract were identified by paper chromatography (Fig. 5). Oligo( [ 14C] ADP-Rib) (I 2,200 cpm corresponding to 6.9 nmole of ADP-Rib) was incubated with 42 pug of glycohydrolase for 7 and 20 min. The reaction was stopped by the addition of Dowex resins and the mixture extracted with 6 M formic acid. About 150 pg of nonradioactive ADP-Rib was added as carrier. The formic acid extract was carefully applied to a sheet of Whatman No. I chromatography paper (50 X 2 cm). The paper was developed overnight in a solvent system as described under Materials and Methods. The chromatogram was dried, cut into 1-cm strips and each strip counted in a standard toluene-based scintillation cocktail. It was found that at 7 and 20 min of incubation the recoveries of the radioactivity with the ADPRib carrier applied to the paper were 87 and 92%, respectively. The identity of radioactive ADP-Rib was also established by using other chromatographic solvent systems (35). The action of venom phosphodiesterase on poly- or oligo(ADP-Rib) yielded two products, namely, AMP and PR-AMP, which can be dephosphorylated completely by E. co/i alkaline phosphatase (19). These dephosphorylated compounds (Ad and R-Ad) are poorly bound to Dowex and can be easily extracted with 0.5 M formic acid (see Fig. 3). On the other hand, ADP-Rib, the product of the glycohydrolase action, cannot be dephosphorylated by alkaline phosphatase and was not extracted from Dowex with 0.5 M formic acid (Fig. 3). These findings were utilized in the present method to distinguish between glycohydrolase and phosphodiesterase activity with oligo(ADP-Rib) as substrate (Table 1). After incubating 25 ,ug of glycohydrolase (with or without E. coli alkaline phosphatase) in the standard assay mixture for 10 min at 37°C the reaction was stopped by the addition of Dowex resins. The mixture was extracted with 0.5 M formic acid. The mixture was centrifuged, the supernatant fluid collected, and the sediment of Dowex extracted with 6 M formic acid. The amount of radioactivity in aliquots of 0.5 M and 6 M formic acid extracts was determined. When treated with glycohydrolase alone, the amount of radioactivity in the 0.5 M formic acid extract was barely detectable while that in the 6 M acid extract was high. This distribution in counts was not affected by prior treatment with E. coli alkaline phosphatase. When similar experiments were carried out with venom phosphodiesterase, about 20% of the radioactivity was extracted with 0.5 M formic acid and the remaining amount with 6 M formic acid. However, when alkaline phosphatase was included in the reaction mixture, most of the radioactivity was extracted with 0.5 M formic acid. Based on these results, the two reaction mixtures, one containing glycohydrolase plus phosphatase and the other containing phosphodiesterase plus phosphatase, were incubated as described above. At the end of the
ASSAY
OF
POLY(ADP-RIB) TABLE
443
GLYCOHYDROLASE I
DETERMINATION OF GLYCOHYDROLASE AND VENOM PHOSPHODIESTERASE ACTIVITIES Activitya (cpm) Enzyme systems __-~ A B C D E F
Control Glycohydrolase Glycohydrolase + alkaline phosphatase Venom phosphodiesterase Venom phosphodiesterase + alkaline phosphatase Combination of C + E
0.5 M” --~ --~ ---_I 88 132 104 428 1759 1642
6.0 M” 49 974 905 1060 141 876
n The composition of the reaction mixture was described in detail under Materials and Methods. The system contained 9,800 cpm of oligo(~C]ADPR) (3.1 nmoks as ADP-Rib) and 19 pg of glycohydrolase or 23 fig of venom phosphodiesterase. In experiments C. E and F. I .5 units of E. c&i alkaline phosphatase were added. The different enzyme systems were incubated for 15 min at 37°C and the reactions were stopped by adding the Dowex suspension. Each mixture was extracted when 0.5 M and 6.0 M formic as indicated in the text. The volume used for the determination of the amount of radioactivity was 0.8 ml. The values shown need to be multiplied by 2.62 to obtain the total amount of radioactivity present in 2.1 ml of each formic acid extract. Experiment A (control) was not incubated and represents the value obtained at time zero. Systems C and E were incubated separately and mixed and the reaction stopped with the Dowex suspension. b Final concentration of formic acid.
incubation period, both reaction mixtures were added to a tube containing 2 ml of Dowex resin suspension. The product of the reaction was extracted sequentiahy with 0.5 and 6 M formic acid as described above. The results as shown in Table 1 indicate that the radioactivity extracted with 0.5 M formic acid corresponded to phosphodiesterase activity (plus phosphatase), while the radioactivity extracted with 6 M formic acid reflected glycohydrolase activity. By the present method, glycohydrolase and phosphodiesterase activities can be distinguished by using a combination of alkaline phosphatase treatment and differential extraction of the reaction products with formic acid. Although the present method of assay is less sensitive than the previously reported procedure of TCA-precipitable poly(ADP-Rib) (13.16), the linearity of the reaction of the latter method was very brief (16), such that the activity of each batch of enzyme preparations had to be adjusted so that not more than 30-40% of the polymer used as substrate was hydrolyzed. In other words, the measured activities varied with the chain length of the polymer when assayed by the latter method. We observed that the activities differed widely when the same enzyme preparation was assayed with polymers containing 20.7 or 35.2 residues of ADP-Rib. These problems were eliminated in the present method. Very reproducible results were obtained when oligomers or polymers were
444
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AND KOIDE
utilized. Moreover, since poly(ADP-Rib) glycohydrolases of calf thymus (16) and of testes (unpublished results) are exoglycohydrolytic enzymes, the present assay method of measuring the production of [‘“Cl ADP-Rib is a more direct determination of the enzymic activity than the TCAprecipitation method. The present method works only for exonucleolytic and not for endonucleolytic cleavage and is less expensive because oligomers can be used as substrates. The principle of the present method can be applied for determinations of other enzymic reactions, providing the substrate and the products of the reaction have significantly different affinities for the resin. In a preliminary study, exploratory experiments were carried out using Dowex 1X-2, chloride form. The method was not feasible because poly(ADPRib) and oligo(ADP-Rib) were poorly bound by the resin in the chloride form. The purification steps for polymers and oligomers of ADP-Rib as outlined in the present paper has the advantages of simplicity, rapidity and good yield, compared to the previously reported methods (12,19). The initial treatment with 0.1 N NaOH greatly improved the yield, and the purity of the final products was equivalent to that obtained by previous methods (12,19). ACKNOWLEDGMENTS The authors express their appreciation to Mrs. Gabriela E. Vizcarra for her excellent technical assistance. This work was partially supported by a grant of the Population Council (No. M73.71).
REFERENCES 1. Chambon, P., Weill, J. D., Doly, J., Strosser, M. T., and Mandei, P. (1966) Biochem. Biophys. Res. Commun. 25, 638443. 2. Nishizuka, Y., Ueda, K., Nakazawa, K., and Hayaishi, 0. (1967)J. Bid. Chem. 242, 3164-3171. 3. Reeder, R. H., Ueda, K., Honjo, T., Nishizuka, Y., and Hayaishi, 0. (1967) 1. Biol. Chem. 242,3 172-3 179. 4. Sugimura, T., Fujimura, S., Hasegawa, S., and Kawamura, Y. (1967) Biochim. Biophys. Acta 138, 438-441. 5. Fujimura, S., Hasegawa, S., Shimizu, Y., and Sugimura, T. (1967) Biochim. Biophys. Acta 145, 247-259. 6. Burzio, L., and Koide, S. S. (1972) FEBS Left. 20, 20-32. 7. Lehmann, A. R., and Shall, S. (1972) FEBS Left. 26, 181-184. 8. Roberts, J. H., Stark, P., and Smulson, M. (1973) Biochem. Biophys. Res. Commun. 52,43-50. 9. Janakidevi, K., and Koh, C. (1974) Biochemistry 13, 1327-1330. 10. Futai, M., and Mizuno, D. (1967)J. Biol. Chem. 242, 5301-5307. 11. Futai, M., Mizuno, D., and Sugimura, T. (1968) j. Biol. Chem. 243, 63254329. 12. Shima, T., Hasegawa, S., Fujimura, S., Matsubara, H., and Sugimura, T. (1969) J. Biol. Chem. 244, 6632-6635. 13. Miwa, M., and Sugimura, T., (1971)5. Eiol. Chem. 246, 6362-6364.
ASSAY OF POLY(ADP-RIB)
GLYCOHYDROLASE
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14. Ueda, D., Oka, J., Narumiya, S., Miyakawa, N., and Hayaishi. 0. (1972) Biochem. Biophys. Res. Commun. 46, 5 16-523. 15. Miyakawa, N., Ueda, K., and Hayaishi, 0. (1972) Biochem. Biophys. Res. Commun. 49, 239-245.
16. Miwa, M., Tanaka, M., Matsushima, T.. and Sugimura, T. (1974) J. Biol. Chem. 249, 3475-3482. 17.
18. 19. 20. 21.
Burzio, L., and Koide, S. S. (1973) Biochem. Biophys. Res. Commtcn. 53, 572-579. Marmur, J. (1961) J. Mol. Biol. 3, 208-218. Sugimura, T., Yoshimura, N., Miwa, M., Nagai, H., and Nago. M. (197 I ) Arch. Biochem. Biophys. 147, 660-665. Shima, T., Fujimura, S., Hasegawa, S., Shimizu, Y.. and Sugimura. T. (1970) J. Bid. Chem. 245, 1327-1330. Lowry. 0. H., Rosebrough, N. J., Farr, A. L.. and Randall. R. J. ( 1951) J. fG(tl. Cl~em. 193, 265-275.