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Degradation of poly(adenosine diphosphate ribose) by homogenates of various normal tissues and tumors of rats
ARCHIVES
OF BIOCHEMISTRY
Degradation Homogenates MASANAO
AND
BIOPHYSICS
167, 54-60 (1975)
of Poly(Adenosine of Various
Normal
Diphosphate
Tissues and Tumors
MIWA, KIMIYO NAKATSUGAWA, MATSUSHIMA, AND TAKASHI
Department of Molecular Tokyo-IO& and Biochemistry
Ribose) by
KAZUKO HARA, SUGIMURA
of Rats’ TAIJIRO
Oncology, The Institute of Medical Science, University of Tokyo, Minato-ku, Division, National Cancer Center Research Institute, Chuo-ku, Tokyo-104, Japan Received July 19, 1974
Poly(ADP-ribose) glycobydrolase, which was first identified in calf thymus and rat liver, was found to be present in all normal rat tissues and tumors. Of the tissues examined, the testis had the highest activity. The major pathway of degradation of poly(ADP-ribose) in all tissues was through poly(ADP-ribose) glycohydrolase. Phosphodiesterase, which was first found in rat liver, was only of minor importance in hydrolysis of poly(ADP-ribose). These findings suggest the importance of poly(ADP-ribose) glycohydrolase both in normal and tumor tissues for the rapid turnover of poly(ADP-ribose).
poly(ADP-Rib), in rat liver. This phosphodiesterase hydrolyzes the pyrophosphate bonds of poly(ADP-Rib) in a fashion of exonuclease (9, lo), to yield mainly with AMP from a terAdo(P)-Rib-(P),2 minus of the chain of poly(ADP-Rib). Recently, we found and purified a new enzyme from calf thymus which hydrolyzes the ribose-ribose bonds of poly(ADP-Rib) in a fashion of exoglycosidase (11, 12). A similar enzyme was also found in rat liver (13, 14) and in L cells (15). The degradation product of poly(ADP-Rib) by this new enzyme was ADP-Rib. This paper describes studies on the degradation of poly(ADP-Rib) in many normal and tumor tissues of rats. Results showed that poly(ADP-Rib) glycohydrolase is more important than phosphodiesterase in biodegradation of poly(ADP-Rib) in all the tissues tested, including tumor tissues. The significance of this finding is discussed.
Poly(ADP-Rib)2 is a biopolymer synthesized by a nuclear enzyme from NAD by polymerization of the ADP-Rib2 moiety of NAD with formation of ribose-ribose (1 + 2) bonds (1). The presence of poly(ADPRib) polymerase in animal cell nuclei is well established (l), and the natural occurrence of the polymer has been suggested by several workers (2-5). At one terminus of its polymer chain poly(ADP-Rib) is covalently bound to histone (6, 7). The biological significance of poly( ADP-Rib) is not yet known although a relationship between poly(ADP-Rib) formation and DNA synthesis was indicated by Burzio and Koide (8), Moreover, the biodegradation of this polymer in various tissues has not yet been fully investigated. Futai et al. (9) first found phosphodiesterase, which hydrolyzes 1 This work was supported by grants from the Ministry of Education, the Society for Promotion of Cancer Research, Waksman Foundation, and the Mitsubishi Foundation. z The abbreviations used are: poly(ADP-Rib), poly(adenosine diphosphate ribose); ADP-Rib, adenosine diphosphate ribose; Ado(P)-Rib-P, 2’(5”-phosphoribosyl)-5’AMP; Ado-Rib-P, 2’-(5”-phosphoribosyl) adenosine; Ado(P)-Rib. 2’-(ribosyl)-5’AMP; AdoRib, 2’-ribosyl adenosine.
MATERIALS
METHODS
Animals. Donryu strain and Buffalo strain rats weighing loo-150 g were used. Animals were killed by decapitation. The liver was quickly perfused with ice-cold saline and removed. The liver and other normal organs were homogenized in 0.25 M sucrose 54
Copyright 0 1975by Academic Press, Inc. All rights of reproduction in any form reserved.
AND
POLY(ADP-RIBOSE) with a Polytron homogenizer running at maximal speed for 1 min. Tumors. Yoshida ascites hepatomas, AH-130, AH-108, AH-7974, and AH-414, and Yoshida sarcoma were transplanted intraperitoneally into Donryu strain rats. Seven days later the cells were harvested, washed with saline, and homogenized as described above. Morris hepatomas, 7316A and 7794A, were transplanted into the thigh muscle of Buffalo strain rats and 2-3 wk and 4-6 wk later, respectively, tumors were removed and homogenized in the same way. Enzymes and chemicals. Poly(ADP-Rib) glycohydrolase was purified from calf thymus as described previously (12). Purified rat liver phosphodiesterase (16) was a generous gift from Dr. M. Futai. Snake venom phosphodiesterase (EC 3.1.4.1) was a product of Worthington Biochemical Corp., Freehold, NJ and traces of contaminating 5’-nucleotidase (EC 3.1.3.5) were removed by the procedure of Sulkowski and Laskowski (17). Escherichia coli alkaline phosphomonoesterase (EC 3.1.3.1) was from Sigma Chemical Co., St. Louis, MO. [Ade-8-“C]Poly(ADP-Rib) (4.3 x 10’ cpm per pmole ADP-Rib residue) was prepared as described previously (18). [“C]Ado(P)-Rib-P and unlabeled Ado(P)-Rib-P were prepared from [“C]poly(ADPRib) and unlabeled poly(ADP-Rib), respectively by the procedures (of Shima et al. (19). [“CIADP-Rib was prepared by hydrolysis of [“C]poly(ADP-Rib) by purified poly(ADP-Rib) glycohydrolase, and then Dowex 1 column chromatography. Coformycin was a generous gift from Dr. H. Umezawa. Adenosine cyclic 3’:5’-monophosphate was purchased from Boehringer Mannheim GmbH, Mannheim, Germany. Assay of degradation of poly(ADP-Rib). Degradation of poly(ADP-Rib) was measured as follows. The incubation mixture contained in 60 ~1, 3 nmoles as ADP-Rib residues of [“C]poly(ADP-Rib), 3 @moles of sodium phosphate buffer (pH 7.5), 0.6 pmole of 2-mercaptoethanol and an appropriate amount of homogenate. In some cases, 0.6 pmole of sodium fluoride or 0.18 Mmole of adenosine cyclic 3’:5’-monophosphate was included in this incubation mixture. After incubatiosn at 37°C for 10 min, 20 gl of the mixture were applied to a filter paper disc to determine the acid-insoluble radioactivity, using Bollum’s procedure (20) with 5% trichloroacetic acid in 0.25% sodium tungstate (pH 2.0). The amount of acid-soluble reaction product increased linearly with time and with the amount of enzyme up to the level of 50% of the original acid-insoluble radioactivity. To analyze the product, the rest of the incubation mixture was heated in a boiling water bath for 2 min. and centrifuged at 10,000 rpm for 5 min. Heating under these conditions did not cause breakdown of poly(ADP-Rib). Then the supernatant, containing more than 95% of the total radioactivity was mixed with authentic markers, and aliquots were subjected
55
GLYCOHYDROLASE
to paper chromatography and thin-layer chromatography. Paper chromatography and cellulose thin-layer chromatography were performed with the following solvent systems. Solvent 1, isobutyric acid-concentrated NH,OH-water, 66:1:33 (v/v/v); solvent 2, 0.1 M sodium phosphate buffer (pH 6.8)-ammonium sulfate-n-propanol, 100:60:2 (v/w/v); solvent 3, water adjusted to pH 10 with NH,OH; solvent 4, n-butanolwater-concentrated NH,OH, 86:14:5 (v/v/v). RESULTS
The activities of the enzyme degrading poly(ADP-Rib) in various normal tissues and transplantable tumors of rats are given in Table I. The testis had the highest degradation activity, expressed per mg of protein. While kidney, thymus, intestinal mucosa, and spleen showed fairly high activities. Other organs had moderate or low activities and the serum had essentially no activity. Rapidly growing Yoshida TABLE
I
DEGRADATION OF PoLY(ADP-Rm BY HOMOGENATES OF VARIOUS NORMAL TISSUES AND TUMORS OF RATS Normal tissue or tumor
Testis Kidney Thymus Intestinal mucosa Spleen Bone marrow Gastric mucosa Liver Lung Brain Heart Pancreas Thigh muscle Yoshida ascites hepatoma AH-7974 AH-130 AH-414 AH-108 Morris hepatoma 7316A 7794A Yoshida sarcoma
Specific activity units”/mg protein
Specific activity units”/mg DNA
4.41 2.21 2.10 1.90 1.78 1.15 1.00 0.85 0.66 0.39 0.24 0.21 0.13
147 83 7 72 23 27 29 73 13 35 52 13 38
1.81 1.46 1.43 1.34
47 26 64 23
0.91 0.93 1.27
47 39 33
4 One unit is defined as the enzyme activity which converts 1 nmole of ADP-Rib residue in poly(ADPRib) to an acid-soluble form per min at 37°C.
56
MIWA ET AL
ascites hepatomas had higher activities per mg of protein than normal liver, while slowly growing Morris hepatomas had almost the same activities as that of normal liver. Activity expressed per mg of DNA was also highest in the testis, and was quite high in the kidney, liver, and intestinal mucosa. Next, the contributions of poly(ADPRib) glycohydrolase and phosphodiesterase to hydrolysis of poly(ADP-Rib) were analyzed. Hydrolysis of [“C ]poly( ADP-Rib) by poly(ADP-Rib) glycohydrolase should yield ADP-Rib (12) while hydrolysis by phosphodiesterase should yield Ado(P)Rib-P from the inner part, with a small amount of AMP from the terminus of poly(ADP-Rib) (10). As shown in Table II, purified poly(ADP-Rib) glycohydrolase
was not inhibited by the presence of 10 mM sodium fluoride, while the purified phosphodiesterase was inhibited 83%. On the contrary, poly(ADP-Rib) glycohydrolase was inhibited 89% by 3 mM adenosine cyclic 3’:5’-monophosphate, while the phosphodiesterase was inhibited only 31%. Therefore, poly(ADP-Rib) glycohydrolase activity in homogenates could be estimated from the rate of poly(ADP-Rib) hydrolysis in the presence of 10 mM sodium fluoride and the phosphodiesterase activity could be roughly estimated by assaying the rate of poly(ADP-Rib) hydrolysis in the presence of adenosine cyclic 3’:5’-monophosphate if poly(ADP-Rib) glycohydrolase activity was not very high. As shown in Table III, the activities for TABLE III
TABLE II SELECTIVE INHIBITIONS BY SODIUM FLUORIDE AND ADENOSINE CYCLIC 3’:5’-MONOPHOSPHATE OF HYDROLYSIS
OF [“C]POL~(ADP-RIB) PURIFIED ENZYMES
Addition
None 10 mM Sodium fluoride 3 mM Adenosine cyclic 3’:5’-monophosphate
Poly(ADPRib) glycohydrolase activity remaining (%P 1OOb loo 11
EFFECTS OF SODIUM FLUORIDE AND ADENOS~NE CYCLIC 3’:5’-MONOPHOSPHATE ON DEGRADATION OF Potv(ADP-Rib) BY HOMOGENATES OF VARIOUSNORMAL TISSUES AND TUMORS OF RATS
BY
Normal tissue or tumor
Remaining activity (%)Owith 10 mM sodium fluoride
Remaining activity (%)” with 3 mM adenosine cyclic 3’:5’-monophosphate
Testis Kidney Thymus Intestinal mucosa Spleen Bone marrow Gastric mucosa Liver Lung Brain Heart Pancreas Thigh muscle Yoshida ascites hepatoma AH-7974 AH-130 AH-414 AH-108 Morris hepatoma 7316A 7794A Yoshida sarcoma
87 92 102 97 97 110 102 88 100 105 96 95 69
41 45 37 36 42 50 39 35 42 56 46 38 39
91 102 108 100
42 40 48 20
100 100 103
67 71 65
Phosphodiesterase activity remaining (WY loo’ 17 69
“The incubation mixture contained in 60 pl, 3 nmoles of [“C]poly(ADP-Rib), 3 Fmoles of sodium phosphate buffer (pH 7.51, 0.6 pmole of 2-mercaptoethanol and 0.57 fig of protein of purified poly(ADPRib) glycohydrolase (121, or 6.8 pg of protein of purified phosphodiesterase (91, with or without 0.6 rmole of sodium fluoride or 0.18 rmole of adenosine cyclic 3’:5’-monophosphate. Mixtures were incubated at 37°C for 10 min for poly(ADP-Rib) glycohydrolase and for 20 min for phosphodiesterase and then heated at 98°C for 2 min and centrifuged. An aliquot of the supernatant was analyzed by paper chromatography with solvent 1 and the extent of hydrolysis was calculated from the radioactivity remaining at the origin. bUnder these conditions 28% of the poly(ADP-Rib) was hydrolyzed and this was taken as 100% activity. c Under these conditions 29% of the poly(ADP-Rib) was hydrolyzed and this was taken as 100%.
DThe specific activity of each organ or tumor listed in Table I was taken as 100%.
POLY(ADP-RIBOSE)
57
GLYCOHYDROLASE
hydrolysis of poly(ADP-Rib) by preparations of all the normal and tumor tissues tested were similar in the presence and absence of 10 mM sodium fluoride. This indicates that poly(ADP-Rib) glycohydrolase is much more important than phosphodiesterase for hydrolysis of poly(ADPRib). This conclusion was supported by the finding that hydrolysis of poly(ADP-Rib) in all normal and tumor tissues tested was inhibited by the presence of 3 mM adenosine cyclic 3’:5’-monophosphate. To confirm this, the products of hydrolysis were analyzed. With all the tissues tested the reaction products were analyzed and some typical results are shown in Fig. 1. Undegraded and partially degraded poly(ADP-Rib) remained at the origin of the paper chromatogram using solvent 1. With preparations of all organs except thigh muscle and pancreas, the major radioactive product was found in the position of ADP-Rib. With the liver preparation, significant radioactivity was found in the position of
ADP-Rib but sometimes more radioactivity was found in the position of hypoxanthine between marker AMP and adenosine. The reaction products were also analyzed by paper chromatography with solvent 2, and results again showed that the major product cochromatographed with marker ADP-Rib. Material in the position of ADPRib on the chromatogram was eluted with water, digested with snake venom phosphodiesterase and analyzed by cellulose thin-layer chromatography using solvent 1. More than 90% of the radioactivity was found in the region of AMP. This confirmed that the radioactivity in the region of ADP-Rib was actually ADP-Rib, not Ado(P)-Rib-P. With preparations of thigh muscle and pancreas, a small amount of radioactivity was found in the region of ADP-Rib. When the reaction was carried out in the presence of 10 mM sodium fluoride to inhibit further degradation of ADP-Rib, much radioactivity was found in this region. The presence of 3 mM adenosine cyclic 3’:5’-monophosphate did not AH- 130
Liver
Distance
from
origin
(cm
-1
1
FIG. 1. Analyses of products of hydrolysis of poly(ADP-Rib) by homogenates of various organs. The incubation mixtures contained in 60 ~1, 3 nmoles of [“C]poly(ADP-Rib), 3 rmoles of sodium phosphate buffer (pH 7.5), 0.6 pmole of 2-mercaptoethanol, and an appropriate amount of tissue homogenate. Mixtures were incubated at 37°C for 10 min and then heated at 98°C for 2 min and centrifuged. The supernatant was mixed with authentic markers and analyzed by paper chromatography with solvent 1 described in the text. Percentages of the radioactivity of both undegraded and partially degraded [“C]poly(ADP-Rib) remaining at the origin are given in the figures.
58
MIWA
completely inhibit glycohydrolase, because the product of hydrolysis with the preparations of testis, liver, and Yoshida ascites hepatoma AH-7974 was proved to be ADPRib, not Ado(P)-Rib-P by cellulose thinlayer chromatography using solvent 1. Namely, it had the same Rf value as ADP-Rib and its hydrolysis by snake venom phosphodiesterase yielded radioactive AMP. These above results show that poly(ADP-Rib) glycohydrolase is present, and is of major importance in the degradation of poly(ADP-Rib) in all the normal and tumor tissues tested. With preparations of liver, kidney, lung, and Morris hepatomas, two radioactive peaks were found besides the main peak of ADP-Rib. These two peaks were investigated with the liver preparation. One peak was found in the region of marker AMP and the other in the region between marker AMP and marker adenosine. These materials were analyzed by cellulose thin layer chromatography using solvents 1, 3, and 4 and Escherichia coli alkaline phosphomonoesterase. Results showed that the former consisted of 80% AMP and 20% inosine and the latter entirely of hypoxanthine. No IMP was found. No appreciable amount of adenosine was found, except on incubation with 20 pg/ml of coformycin, a potent inhibitor for adenosine deaminase (21). To study the origins of these minor reaction products, the time-dependent changes in these products were studied using liver homogenate. In this experiment the extent of hydrolysis was determined by measuring the amount of radioactivity remaining at the origin of the paper chromatogram. After 5 min, when 15% of the poly(ADP-Rib) had been hydrolyzed, the main radioactive product was found in the region of ADP-Rib. After 10 min, when 31% of the poly(ADP-Rib) had been hydrolyzed, three peaks were found in the regions of ADP-Rib, AMP and hypoxanthine, in the order of decreasing magnitude. After 20 min, when 60% of poly(ADPRib) had been hydrolyzed, the same peaks were found as after 10 min, but their orders of magnitude were reversed. On incubation with 10 mM sodium fluoride for 10 min, the peaks in the regions of hypoxanthine and
ET AL.
AMP were much smaller, with a comparable increase in the region of ADP-Rib without any change in the percentage hydrolysis of poly(ADP-Rib) compared to that in the absence of sodium fluoride. These experiments suggest that AMP, inosine, and hypoxanthine might be further degradation products of ADP-Rib. To examine the metabolic fates of ADPRib and Ado(P)-Rib-P, labeled samples were incubated with the liver homogenate for 10 min and the products were analyzed with paper chromatography. As shown in Fig. 2, [“C]ADP-Rib was hydrolyzed yielding several radioactive peaks, including those in the regions of AMP and hypoxanthine. The pattern was similar to that obtained on incubation of [“C]poly(ADPRib) with the liver homogenate except for the absence of undegraded poly(ADP-Rib) at the origin. [l’C]Ado(P)-Rib-P was not hydrolyzed under these conditions, even on incubation for 20 min. Thus, under these conditions, neither Ado-Rib-P, Ado(P)Rib, nor Ado-Rib were formed from Ado(P)-Rib-P. These results also indicate that poly(ADP-Rib) was mainly hydrolyzed through ADP-Rib with poly(ADPRib) glycohydrolase, not through Ado(P)Rib-P with phosphodiesterase in the liver homogenate. The above results are all compatible with the conclusion that poly(ADP-Rib) glycohydrolase is the main enzyme involved in hydrolysis of poly(ADP-Rib) in these tissues. DISCUSSION
Poly(ADP-Rib) glycohydrolase has been found in calf thymus (11, 12), rat liver (13, 14), and L cells (15). In this work, we demonstrated the presence of poly(ADPRib) glycohydrolase in most normal and malignant tissues of rats, as shown in Tables I and III, and Fig. 1. We found that poly(ADP-Rib) glycohydrolase is more important than phosphodiesterase (9) in degradation of poly(ADP-Rib) in these tissues. The reported K, values of 0.54 pM and 28 PM for poly( ADP-Rib) glycohydrolase (12) and phosphodiesterase (9), respectively, are compatible with these findings. The product of poly(ADP-Rib) glycohy-
POLY(ADP-RIBOSE)
59
GLYCOHYDROLASE
600.
@C”ClPoly(ADP-Rib)
400.
1 .o L
~ADWbDW-MPA
F,oi’
A$
zoo-
u) I
E 0 \ E
O
i
ADP-Rib
600
@“Cl
4
ADP-RI
b
2 400 AMP 4
200
Ad0 4
,x .-
.-> ::
2 ‘p rr”
4
J-L
0
Ado(P)-Rib-P
600
Ado(P)-Rib-P
400. Ado 4
200.
4 0 LO
40
Disfonce
from
origin
(cm)
FIG. 2. Metabolisms of poly(ADP-Rib), ADP-Rib, and Ado(P)-Rib-P, by liver homogenate. The incubation mixture contained in 66 ~1, 3 pmoles of sodium phosphate buffer (pH 7.5), 0.6 pmole of 2-mercaptoethanol, 0.2 mg of protein of liver homogenate with the various substrates indicated; (1) 2.5 nmoles of [“C]poly(ADP-Rib), (2) 3.J nmoles of [“CIADP-Rib, and (3) 2.6 nmoles of [“C]Ado(P)-Rib-P. Mixtures were incubated at 37°C for 10 min and the products were analyzed as described under Materials and Methods. Solvent 1 was used for paper chromatography.
drolase, ADP-Rib, was rapidly hydrolyzed in a liver homogenate, while the product of phosphodiesterase, Ado(P)-Rib-P, was not metabolized further as shown in Fig. 2. This is reasonable since poly(ADP-Rib) is mainly degraded by poly(ADP-Rib) glycohydrolase forming ADP-Rib. With liver homogenate, poly(ADP-Rib) was found to be metabolized to ADP-Rib, AMP, adenosine, inosine., and hypoxanthine, in this order. It is noteworthy that testis had the highest specific activity of poly(ADP-Rib) glycohydrolase, as shown in Table I and Fig. 1. It is of interest that the poly(ADPRib) glycohydrolase activity, expressed per
mg of protein, was a little higher in rapidly growing Yoshida ascites hepatomas and Yoshida sarcoma than in normal liver, while in slowly growing Morris hepatomas, the activity was essentially the same as that in normal liver. However, this difference was not apparent when the activity was expressed per mg of DNA. The significance of these findings in connection with the metabolism of poly(ADP-Rib) in the nucleus remains to be elucidated. The natural occurrence of poly(ADPRib) has been reported (24, but its concentration seems to be very low. This might be related to the relatively rapid turnover of poly(ADP-Rib) due to the ac-
60
MIWA
tion of poly(ADP-Rib) glycohydrolase. Gill reported the poly(ADP-Rib)-synthesizing activities of high salt extracts of various rat organs and found that the half-life of poly(ADP-Rib) in rat liver extract was about 5 min (22). When poly(ADP-Rib) is partially hydrolyzed by phosphodiesterase, new chain elongation of the partially hydrolyzed product, namely, ADP-ribosylation, does not seem to be possible. However, when poly(ADP-Rib) is partially hydrolyzed by poly(ADP-Rib) glycohydrolase, reelongation of the chain of the partially hydrolyzed product by ADP-ribosylation may be possible (12). In this sense, the action of poly(ADP-Rib) glycohydrolase seems to be more physiological than that of phosphodiesterase. REFERENCES 1. SUGIMURA, T. (1973) in Progress in Nucleic Acid Research and Molecular Biology, (Davidson, J. N., and Cohn, W. E., eds.), Vol. 13, pp. 127-151, Academic Press, New York. 2. DOLY, J., AND MANDEL, P. (1967) C. R. Acad. Sci. Paris 264, 2687-2690. 3. HILZ, H. BREDEHORST,R., NOLDE, S., ANDKI~TLER, M. (1972) 2. Physiol. Chem. 353,&W-849. 4. COLYER, R. A., BLIRDETTE,K. E., ANDKIDWELL, W. R. (1973) Biochem. Biophys. Res. Commun. 53, 960466. 5. SMITH, J. A., AND STOCKEN, L. A. (1973) Biochem. Biophys. Res. Commun. 54,297-300. 6. NISHIZUKA, Y., UEDA, K., HONJO, T., AND HAYAISHI, 0. (1968) J. Biol. Chem. 243,3765-3767.
ETAL. 7. OTAKE, H., MIWA, M., FUJIMURA, S., AND SUGIMLIRA,T. (1969) J. Biochem. 65, 145-146. 8. BURZIO, L., AND KOIDE, S. S. (1970) Biochem. Biophys. Res. Commun. 40, 1013-1020. 9. FUTAI, M., MIZUNO, D., AND SUGIMURA,T. (1968) J. Biol. Chem. 243,6325-6329. 10. MATSUBARA, H., HASEGAWA, S., FUJIMURA, S., SHIMA, T., SUGIMURA, T., AND FUTAI, M. (1970) J. Biol. Chem. 245, 4317-4320. 11. MIWA, M., AND SUGIMURA,T. (1971) J. Biol. Chem. 246, 63624364. 12. MIWA, M., TANAKA, M., MATSUSHIMA, T., AND SUGIMURA, T. (1974) J. Biol. Chem. 249, 3475-3482. 13. UEDA, K., OKA, J., NARUMIYA, S., MIYAKAWA, N., AND HAYAISHI, 0. (1972) Biochem. Biophys. Res. Commun. 46, 516-523. 14. MIYAKAWA, N., UEDA, K., ANDHAYAISHI, 0. (1972) Biochem. Biophys. Res. Commun. 49,239-245. 15. STONE, P. R., WHISH, W. J. D., AND SHALL, S. (1973) Fed. Eur. Biochem. Sot. Lett. 36, 334-338. 16. FUTAI, M., AND MIZUNO, D. (1967) J. Biol. Chem. 242, 5301-5307. 17. SULKOWSKI, E., AND LASKOWSKI, M. (1971) Biochim. Biophys. Acta 240. 443-447. 18. SUGIMURA, T., YOSHIMURA, N., MIWA, M., NAGAI, H., AND NAGAO, M. (1971) Arch. Biochem. Biophys. 147, 660-665. 19. SHIMA, T., HASEGAWA, S., FUJIMURA, S., MATSUBARA, H., AND SUGIMUR~ T. (1969) J. Biol. Chem. 244.6632-6635. 20. BOLLUM, F. J. (1966) in Procedures in Nucleic Acid Research (Cantoni, G. L., and Davies, D. R. eds.), pp. 296-300, Harper and Row, New York. 21. SAWA, T., FUKAGAWA,Y., HOMMA, I., TAKEUCHI, T., AND UMEZAWA, H. (1967) J. Antibiot. 20, 227-231. 22. GILL, D. M. (1972) J. Biol. Chem. 247,5964-5971.
OF BIOCHEMISTRY
Degradation Homogenates MASANAO
AND
BIOPHYSICS
167, 54-60 (1975)
of Poly(Adenosine of Various
Normal
Diphosphate
Tissues and Tumors
MIWA, KIMIYO NAKATSUGAWA, MATSUSHIMA, AND TAKASHI
Department of Molecular Tokyo-IO& and Biochemistry
Ribose) by
KAZUKO HARA, SUGIMURA
of Rats’ TAIJIRO
Oncology, The Institute of Medical Science, University of Tokyo, Minato-ku, Division, National Cancer Center Research Institute, Chuo-ku, Tokyo-104, Japan Received July 19, 1974
Poly(ADP-ribose) glycobydrolase, which was first identified in calf thymus and rat liver, was found to be present in all normal rat tissues and tumors. Of the tissues examined, the testis had the highest activity. The major pathway of degradation of poly(ADP-ribose) in all tissues was through poly(ADP-ribose) glycohydrolase. Phosphodiesterase, which was first found in rat liver, was only of minor importance in hydrolysis of poly(ADP-ribose). These findings suggest the importance of poly(ADP-ribose) glycohydrolase both in normal and tumor tissues for the rapid turnover of poly(ADP-ribose).
poly(ADP-Rib), in rat liver. This phosphodiesterase hydrolyzes the pyrophosphate bonds of poly(ADP-Rib) in a fashion of exonuclease (9, lo), to yield mainly with AMP from a terAdo(P)-Rib-(P),2 minus of the chain of poly(ADP-Rib). Recently, we found and purified a new enzyme from calf thymus which hydrolyzes the ribose-ribose bonds of poly(ADP-Rib) in a fashion of exoglycosidase (11, 12). A similar enzyme was also found in rat liver (13, 14) and in L cells (15). The degradation product of poly(ADP-Rib) by this new enzyme was ADP-Rib. This paper describes studies on the degradation of poly(ADP-Rib) in many normal and tumor tissues of rats. Results showed that poly(ADP-Rib) glycohydrolase is more important than phosphodiesterase in biodegradation of poly(ADP-Rib) in all the tissues tested, including tumor tissues. The significance of this finding is discussed.
Poly(ADP-Rib)2 is a biopolymer synthesized by a nuclear enzyme from NAD by polymerization of the ADP-Rib2 moiety of NAD with formation of ribose-ribose (1 + 2) bonds (1). The presence of poly(ADPRib) polymerase in animal cell nuclei is well established (l), and the natural occurrence of the polymer has been suggested by several workers (2-5). At one terminus of its polymer chain poly(ADP-Rib) is covalently bound to histone (6, 7). The biological significance of poly( ADP-Rib) is not yet known although a relationship between poly(ADP-Rib) formation and DNA synthesis was indicated by Burzio and Koide (8), Moreover, the biodegradation of this polymer in various tissues has not yet been fully investigated. Futai et al. (9) first found phosphodiesterase, which hydrolyzes 1 This work was supported by grants from the Ministry of Education, the Society for Promotion of Cancer Research, Waksman Foundation, and the Mitsubishi Foundation. z The abbreviations used are: poly(ADP-Rib), poly(adenosine diphosphate ribose); ADP-Rib, adenosine diphosphate ribose; Ado(P)-Rib-P, 2’(5”-phosphoribosyl)-5’AMP; Ado-Rib-P, 2’-(5”-phosphoribosyl) adenosine; Ado(P)-Rib. 2’-(ribosyl)-5’AMP; AdoRib, 2’-ribosyl adenosine.
MATERIALS
METHODS
Animals. Donryu strain and Buffalo strain rats weighing loo-150 g were used. Animals were killed by decapitation. The liver was quickly perfused with ice-cold saline and removed. The liver and other normal organs were homogenized in 0.25 M sucrose 54
Copyright 0 1975by Academic Press, Inc. All rights of reproduction in any form reserved.
AND
POLY(ADP-RIBOSE) with a Polytron homogenizer running at maximal speed for 1 min. Tumors. Yoshida ascites hepatomas, AH-130, AH-108, AH-7974, and AH-414, and Yoshida sarcoma were transplanted intraperitoneally into Donryu strain rats. Seven days later the cells were harvested, washed with saline, and homogenized as described above. Morris hepatomas, 7316A and 7794A, were transplanted into the thigh muscle of Buffalo strain rats and 2-3 wk and 4-6 wk later, respectively, tumors were removed and homogenized in the same way. Enzymes and chemicals. Poly(ADP-Rib) glycohydrolase was purified from calf thymus as described previously (12). Purified rat liver phosphodiesterase (16) was a generous gift from Dr. M. Futai. Snake venom phosphodiesterase (EC 3.1.4.1) was a product of Worthington Biochemical Corp., Freehold, NJ and traces of contaminating 5’-nucleotidase (EC 3.1.3.5) were removed by the procedure of Sulkowski and Laskowski (17). Escherichia coli alkaline phosphomonoesterase (EC 3.1.3.1) was from Sigma Chemical Co., St. Louis, MO. [Ade-8-“C]Poly(ADP-Rib) (4.3 x 10’ cpm per pmole ADP-Rib residue) was prepared as described previously (18). [“C]Ado(P)-Rib-P and unlabeled Ado(P)-Rib-P were prepared from [“C]poly(ADPRib) and unlabeled poly(ADP-Rib), respectively by the procedures (of Shima et al. (19). [“CIADP-Rib was prepared by hydrolysis of [“C]poly(ADP-Rib) by purified poly(ADP-Rib) glycohydrolase, and then Dowex 1 column chromatography. Coformycin was a generous gift from Dr. H. Umezawa. Adenosine cyclic 3’:5’-monophosphate was purchased from Boehringer Mannheim GmbH, Mannheim, Germany. Assay of degradation of poly(ADP-Rib). Degradation of poly(ADP-Rib) was measured as follows. The incubation mixture contained in 60 ~1, 3 nmoles as ADP-Rib residues of [“C]poly(ADP-Rib), 3 @moles of sodium phosphate buffer (pH 7.5), 0.6 pmole of 2-mercaptoethanol and an appropriate amount of homogenate. In some cases, 0.6 pmole of sodium fluoride or 0.18 Mmole of adenosine cyclic 3’:5’-monophosphate was included in this incubation mixture. After incubatiosn at 37°C for 10 min, 20 gl of the mixture were applied to a filter paper disc to determine the acid-insoluble radioactivity, using Bollum’s procedure (20) with 5% trichloroacetic acid in 0.25% sodium tungstate (pH 2.0). The amount of acid-soluble reaction product increased linearly with time and with the amount of enzyme up to the level of 50% of the original acid-insoluble radioactivity. To analyze the product, the rest of the incubation mixture was heated in a boiling water bath for 2 min. and centrifuged at 10,000 rpm for 5 min. Heating under these conditions did not cause breakdown of poly(ADP-Rib). Then the supernatant, containing more than 95% of the total radioactivity was mixed with authentic markers, and aliquots were subjected
55
GLYCOHYDROLASE
to paper chromatography and thin-layer chromatography. Paper chromatography and cellulose thin-layer chromatography were performed with the following solvent systems. Solvent 1, isobutyric acid-concentrated NH,OH-water, 66:1:33 (v/v/v); solvent 2, 0.1 M sodium phosphate buffer (pH 6.8)-ammonium sulfate-n-propanol, 100:60:2 (v/w/v); solvent 3, water adjusted to pH 10 with NH,OH; solvent 4, n-butanolwater-concentrated NH,OH, 86:14:5 (v/v/v). RESULTS
The activities of the enzyme degrading poly(ADP-Rib) in various normal tissues and transplantable tumors of rats are given in Table I. The testis had the highest degradation activity, expressed per mg of protein. While kidney, thymus, intestinal mucosa, and spleen showed fairly high activities. Other organs had moderate or low activities and the serum had essentially no activity. Rapidly growing Yoshida TABLE
I
DEGRADATION OF PoLY(ADP-Rm BY HOMOGENATES OF VARIOUS NORMAL TISSUES AND TUMORS OF RATS Normal tissue or tumor
Testis Kidney Thymus Intestinal mucosa Spleen Bone marrow Gastric mucosa Liver Lung Brain Heart Pancreas Thigh muscle Yoshida ascites hepatoma AH-7974 AH-130 AH-414 AH-108 Morris hepatoma 7316A 7794A Yoshida sarcoma
Specific activity units”/mg protein
Specific activity units”/mg DNA
4.41 2.21 2.10 1.90 1.78 1.15 1.00 0.85 0.66 0.39 0.24 0.21 0.13
147 83 7 72 23 27 29 73 13 35 52 13 38
1.81 1.46 1.43 1.34
47 26 64 23
0.91 0.93 1.27
47 39 33
4 One unit is defined as the enzyme activity which converts 1 nmole of ADP-Rib residue in poly(ADPRib) to an acid-soluble form per min at 37°C.
56
MIWA ET AL
ascites hepatomas had higher activities per mg of protein than normal liver, while slowly growing Morris hepatomas had almost the same activities as that of normal liver. Activity expressed per mg of DNA was also highest in the testis, and was quite high in the kidney, liver, and intestinal mucosa. Next, the contributions of poly(ADPRib) glycohydrolase and phosphodiesterase to hydrolysis of poly(ADP-Rib) were analyzed. Hydrolysis of [“C ]poly( ADP-Rib) by poly(ADP-Rib) glycohydrolase should yield ADP-Rib (12) while hydrolysis by phosphodiesterase should yield Ado(P)Rib-P from the inner part, with a small amount of AMP from the terminus of poly(ADP-Rib) (10). As shown in Table II, purified poly(ADP-Rib) glycohydrolase
was not inhibited by the presence of 10 mM sodium fluoride, while the purified phosphodiesterase was inhibited 83%. On the contrary, poly(ADP-Rib) glycohydrolase was inhibited 89% by 3 mM adenosine cyclic 3’:5’-monophosphate, while the phosphodiesterase was inhibited only 31%. Therefore, poly(ADP-Rib) glycohydrolase activity in homogenates could be estimated from the rate of poly(ADP-Rib) hydrolysis in the presence of 10 mM sodium fluoride and the phosphodiesterase activity could be roughly estimated by assaying the rate of poly(ADP-Rib) hydrolysis in the presence of adenosine cyclic 3’:5’-monophosphate if poly(ADP-Rib) glycohydrolase activity was not very high. As shown in Table III, the activities for TABLE III
TABLE II SELECTIVE INHIBITIONS BY SODIUM FLUORIDE AND ADENOSINE CYCLIC 3’:5’-MONOPHOSPHATE OF HYDROLYSIS
OF [“C]POL~(ADP-RIB) PURIFIED ENZYMES
Addition
None 10 mM Sodium fluoride 3 mM Adenosine cyclic 3’:5’-monophosphate
Poly(ADPRib) glycohydrolase activity remaining (%P 1OOb loo 11
EFFECTS OF SODIUM FLUORIDE AND ADENOS~NE CYCLIC 3’:5’-MONOPHOSPHATE ON DEGRADATION OF Potv(ADP-Rib) BY HOMOGENATES OF VARIOUSNORMAL TISSUES AND TUMORS OF RATS
BY
Normal tissue or tumor
Remaining activity (%)Owith 10 mM sodium fluoride
Remaining activity (%)” with 3 mM adenosine cyclic 3’:5’-monophosphate
Testis Kidney Thymus Intestinal mucosa Spleen Bone marrow Gastric mucosa Liver Lung Brain Heart Pancreas Thigh muscle Yoshida ascites hepatoma AH-7974 AH-130 AH-414 AH-108 Morris hepatoma 7316A 7794A Yoshida sarcoma
87 92 102 97 97 110 102 88 100 105 96 95 69
41 45 37 36 42 50 39 35 42 56 46 38 39
91 102 108 100
42 40 48 20
100 100 103
67 71 65
Phosphodiesterase activity remaining (WY loo’ 17 69
“The incubation mixture contained in 60 pl, 3 nmoles of [“C]poly(ADP-Rib), 3 Fmoles of sodium phosphate buffer (pH 7.51, 0.6 pmole of 2-mercaptoethanol and 0.57 fig of protein of purified poly(ADPRib) glycohydrolase (121, or 6.8 pg of protein of purified phosphodiesterase (91, with or without 0.6 rmole of sodium fluoride or 0.18 rmole of adenosine cyclic 3’:5’-monophosphate. Mixtures were incubated at 37°C for 10 min for poly(ADP-Rib) glycohydrolase and for 20 min for phosphodiesterase and then heated at 98°C for 2 min and centrifuged. An aliquot of the supernatant was analyzed by paper chromatography with solvent 1 and the extent of hydrolysis was calculated from the radioactivity remaining at the origin. bUnder these conditions 28% of the poly(ADP-Rib) was hydrolyzed and this was taken as 100% activity. c Under these conditions 29% of the poly(ADP-Rib) was hydrolyzed and this was taken as 100%.
DThe specific activity of each organ or tumor listed in Table I was taken as 100%.
POLY(ADP-RIBOSE)
57
GLYCOHYDROLASE
hydrolysis of poly(ADP-Rib) by preparations of all the normal and tumor tissues tested were similar in the presence and absence of 10 mM sodium fluoride. This indicates that poly(ADP-Rib) glycohydrolase is much more important than phosphodiesterase for hydrolysis of poly(ADPRib). This conclusion was supported by the finding that hydrolysis of poly(ADP-Rib) in all normal and tumor tissues tested was inhibited by the presence of 3 mM adenosine cyclic 3’:5’-monophosphate. To confirm this, the products of hydrolysis were analyzed. With all the tissues tested the reaction products were analyzed and some typical results are shown in Fig. 1. Undegraded and partially degraded poly(ADP-Rib) remained at the origin of the paper chromatogram using solvent 1. With preparations of all organs except thigh muscle and pancreas, the major radioactive product was found in the position of ADP-Rib. With the liver preparation, significant radioactivity was found in the position of
ADP-Rib but sometimes more radioactivity was found in the position of hypoxanthine between marker AMP and adenosine. The reaction products were also analyzed by paper chromatography with solvent 2, and results again showed that the major product cochromatographed with marker ADP-Rib. Material in the position of ADPRib on the chromatogram was eluted with water, digested with snake venom phosphodiesterase and analyzed by cellulose thin-layer chromatography using solvent 1. More than 90% of the radioactivity was found in the region of AMP. This confirmed that the radioactivity in the region of ADP-Rib was actually ADP-Rib, not Ado(P)-Rib-P. With preparations of thigh muscle and pancreas, a small amount of radioactivity was found in the region of ADP-Rib. When the reaction was carried out in the presence of 10 mM sodium fluoride to inhibit further degradation of ADP-Rib, much radioactivity was found in this region. The presence of 3 mM adenosine cyclic 3’:5’-monophosphate did not AH- 130
Liver
Distance
from
origin
(cm
-1
1
FIG. 1. Analyses of products of hydrolysis of poly(ADP-Rib) by homogenates of various organs. The incubation mixtures contained in 60 ~1, 3 nmoles of [“C]poly(ADP-Rib), 3 rmoles of sodium phosphate buffer (pH 7.5), 0.6 pmole of 2-mercaptoethanol, and an appropriate amount of tissue homogenate. Mixtures were incubated at 37°C for 10 min and then heated at 98°C for 2 min and centrifuged. The supernatant was mixed with authentic markers and analyzed by paper chromatography with solvent 1 described in the text. Percentages of the radioactivity of both undegraded and partially degraded [“C]poly(ADP-Rib) remaining at the origin are given in the figures.
58
MIWA
completely inhibit glycohydrolase, because the product of hydrolysis with the preparations of testis, liver, and Yoshida ascites hepatoma AH-7974 was proved to be ADPRib, not Ado(P)-Rib-P by cellulose thinlayer chromatography using solvent 1. Namely, it had the same Rf value as ADP-Rib and its hydrolysis by snake venom phosphodiesterase yielded radioactive AMP. These above results show that poly(ADP-Rib) glycohydrolase is present, and is of major importance in the degradation of poly(ADP-Rib) in all the normal and tumor tissues tested. With preparations of liver, kidney, lung, and Morris hepatomas, two radioactive peaks were found besides the main peak of ADP-Rib. These two peaks were investigated with the liver preparation. One peak was found in the region of marker AMP and the other in the region between marker AMP and marker adenosine. These materials were analyzed by cellulose thin layer chromatography using solvents 1, 3, and 4 and Escherichia coli alkaline phosphomonoesterase. Results showed that the former consisted of 80% AMP and 20% inosine and the latter entirely of hypoxanthine. No IMP was found. No appreciable amount of adenosine was found, except on incubation with 20 pg/ml of coformycin, a potent inhibitor for adenosine deaminase (21). To study the origins of these minor reaction products, the time-dependent changes in these products were studied using liver homogenate. In this experiment the extent of hydrolysis was determined by measuring the amount of radioactivity remaining at the origin of the paper chromatogram. After 5 min, when 15% of the poly(ADP-Rib) had been hydrolyzed, the main radioactive product was found in the region of ADP-Rib. After 10 min, when 31% of the poly(ADP-Rib) had been hydrolyzed, three peaks were found in the regions of ADP-Rib, AMP and hypoxanthine, in the order of decreasing magnitude. After 20 min, when 60% of poly(ADPRib) had been hydrolyzed, the same peaks were found as after 10 min, but their orders of magnitude were reversed. On incubation with 10 mM sodium fluoride for 10 min, the peaks in the regions of hypoxanthine and
ET AL.
AMP were much smaller, with a comparable increase in the region of ADP-Rib without any change in the percentage hydrolysis of poly(ADP-Rib) compared to that in the absence of sodium fluoride. These experiments suggest that AMP, inosine, and hypoxanthine might be further degradation products of ADP-Rib. To examine the metabolic fates of ADPRib and Ado(P)-Rib-P, labeled samples were incubated with the liver homogenate for 10 min and the products were analyzed with paper chromatography. As shown in Fig. 2, [“C]ADP-Rib was hydrolyzed yielding several radioactive peaks, including those in the regions of AMP and hypoxanthine. The pattern was similar to that obtained on incubation of [“C]poly(ADPRib) with the liver homogenate except for the absence of undegraded poly(ADP-Rib) at the origin. [l’C]Ado(P)-Rib-P was not hydrolyzed under these conditions, even on incubation for 20 min. Thus, under these conditions, neither Ado-Rib-P, Ado(P)Rib, nor Ado-Rib were formed from Ado(P)-Rib-P. These results also indicate that poly(ADP-Rib) was mainly hydrolyzed through ADP-Rib with poly(ADPRib) glycohydrolase, not through Ado(P)Rib-P with phosphodiesterase in the liver homogenate. The above results are all compatible with the conclusion that poly(ADP-Rib) glycohydrolase is the main enzyme involved in hydrolysis of poly(ADP-Rib) in these tissues. DISCUSSION
Poly(ADP-Rib) glycohydrolase has been found in calf thymus (11, 12), rat liver (13, 14), and L cells (15). In this work, we demonstrated the presence of poly(ADPRib) glycohydrolase in most normal and malignant tissues of rats, as shown in Tables I and III, and Fig. 1. We found that poly(ADP-Rib) glycohydrolase is more important than phosphodiesterase (9) in degradation of poly(ADP-Rib) in these tissues. The reported K, values of 0.54 pM and 28 PM for poly( ADP-Rib) glycohydrolase (12) and phosphodiesterase (9), respectively, are compatible with these findings. The product of poly(ADP-Rib) glycohy-
POLY(ADP-RIBOSE)
59
GLYCOHYDROLASE
600.
@C”ClPoly(ADP-Rib)
400.
1 .o L
~ADWbDW-MPA
F,oi’
A$
zoo-
u) I
E 0 \ E
O
i
ADP-Rib
600
@“Cl
4
ADP-RI
b
2 400 AMP 4
200
Ad0 4
,x .-
.-> ::
2 ‘p rr”
4
J-L
0
Ado(P)-Rib-P
600
Ado(P)-Rib-P
400. Ado 4
200.
4 0 LO
40
Disfonce
from
origin
(cm)
FIG. 2. Metabolisms of poly(ADP-Rib), ADP-Rib, and Ado(P)-Rib-P, by liver homogenate. The incubation mixture contained in 66 ~1, 3 pmoles of sodium phosphate buffer (pH 7.5), 0.6 pmole of 2-mercaptoethanol, 0.2 mg of protein of liver homogenate with the various substrates indicated; (1) 2.5 nmoles of [“C]poly(ADP-Rib), (2) 3.J nmoles of [“CIADP-Rib, and (3) 2.6 nmoles of [“C]Ado(P)-Rib-P. Mixtures were incubated at 37°C for 10 min and the products were analyzed as described under Materials and Methods. Solvent 1 was used for paper chromatography.
drolase, ADP-Rib, was rapidly hydrolyzed in a liver homogenate, while the product of phosphodiesterase, Ado(P)-Rib-P, was not metabolized further as shown in Fig. 2. This is reasonable since poly(ADP-Rib) is mainly degraded by poly(ADP-Rib) glycohydrolase forming ADP-Rib. With liver homogenate, poly(ADP-Rib) was found to be metabolized to ADP-Rib, AMP, adenosine, inosine., and hypoxanthine, in this order. It is noteworthy that testis had the highest specific activity of poly(ADP-Rib) glycohydrolase, as shown in Table I and Fig. 1. It is of interest that the poly(ADPRib) glycohydrolase activity, expressed per
mg of protein, was a little higher in rapidly growing Yoshida ascites hepatomas and Yoshida sarcoma than in normal liver, while in slowly growing Morris hepatomas, the activity was essentially the same as that in normal liver. However, this difference was not apparent when the activity was expressed per mg of DNA. The significance of these findings in connection with the metabolism of poly(ADP-Rib) in the nucleus remains to be elucidated. The natural occurrence of poly(ADPRib) has been reported (24, but its concentration seems to be very low. This might be related to the relatively rapid turnover of poly(ADP-Rib) due to the ac-
60
MIWA
tion of poly(ADP-Rib) glycohydrolase. Gill reported the poly(ADP-Rib)-synthesizing activities of high salt extracts of various rat organs and found that the half-life of poly(ADP-Rib) in rat liver extract was about 5 min (22). When poly(ADP-Rib) is partially hydrolyzed by phosphodiesterase, new chain elongation of the partially hydrolyzed product, namely, ADP-ribosylation, does not seem to be possible. However, when poly(ADP-Rib) is partially hydrolyzed by poly(ADP-Rib) glycohydrolase, reelongation of the chain of the partially hydrolyzed product by ADP-ribosylation may be possible (12). In this sense, the action of poly(ADP-Rib) glycohydrolase seems to be more physiological than that of phosphodiesterase. REFERENCES 1. SUGIMURA, T. (1973) in Progress in Nucleic Acid Research and Molecular Biology, (Davidson, J. N., and Cohn, W. E., eds.), Vol. 13, pp. 127-151, Academic Press, New York. 2. DOLY, J., AND MANDEL, P. (1967) C. R. Acad. Sci. Paris 264, 2687-2690. 3. HILZ, H. BREDEHORST,R., NOLDE, S., ANDKI~TLER, M. (1972) 2. Physiol. Chem. 353,&W-849. 4. COLYER, R. A., BLIRDETTE,K. E., ANDKIDWELL, W. R. (1973) Biochem. Biophys. Res. Commun. 53, 960466. 5. SMITH, J. A., AND STOCKEN, L. A. (1973) Biochem. Biophys. Res. Commun. 54,297-300. 6. NISHIZUKA, Y., UEDA, K., HONJO, T., AND HAYAISHI, 0. (1968) J. Biol. Chem. 243,3765-3767.
ETAL. 7. OTAKE, H., MIWA, M., FUJIMURA, S., AND SUGIMLIRA,T. (1969) J. Biochem. 65, 145-146. 8. BURZIO, L., AND KOIDE, S. S. (1970) Biochem. Biophys. Res. Commun. 40, 1013-1020. 9. FUTAI, M., MIZUNO, D., AND SUGIMURA,T. (1968) J. Biol. Chem. 243,6325-6329. 10. MATSUBARA, H., HASEGAWA, S., FUJIMURA, S., SHIMA, T., SUGIMURA, T., AND FUTAI, M. (1970) J. Biol. Chem. 245, 4317-4320. 11. MIWA, M., AND SUGIMURA,T. (1971) J. Biol. Chem. 246, 63624364. 12. MIWA, M., TANAKA, M., MATSUSHIMA, T., AND SUGIMURA, T. (1974) J. Biol. Chem. 249, 3475-3482. 13. UEDA, K., OKA, J., NARUMIYA, S., MIYAKAWA, N., AND HAYAISHI, 0. (1972) Biochem. Biophys. Res. Commun. 46, 516-523. 14. MIYAKAWA, N., UEDA, K., ANDHAYAISHI, 0. (1972) Biochem. Biophys. Res. Commun. 49,239-245. 15. STONE, P. R., WHISH, W. J. D., AND SHALL, S. (1973) Fed. Eur. Biochem. Sot. Lett. 36, 334-338. 16. FUTAI, M., AND MIZUNO, D. (1967) J. Biol. Chem. 242, 5301-5307. 17. SULKOWSKI, E., AND LASKOWSKI, M. (1971) Biochim. Biophys. Acta 240. 443-447. 18. SUGIMURA, T., YOSHIMURA, N., MIWA, M., NAGAI, H., AND NAGAO, M. (1971) Arch. Biochem. Biophys. 147, 660-665. 19. SHIMA, T., HASEGAWA, S., FUJIMURA, S., MATSUBARA, H., AND SUGIMUR~ T. (1969) J. Biol. Chem. 244.6632-6635. 20. BOLLUM, F. J. (1966) in Procedures in Nucleic Acid Research (Cantoni, G. L., and Davies, D. R. eds.), pp. 296-300, Harper and Row, New York. 21. SAWA, T., FUKAGAWA,Y., HOMMA, I., TAKEUCHI, T., AND UMEZAWA, H. (1967) J. Antibiot. 20, 227-231. 22. GILL, D. M. (1972) J. Biol. Chem. 247,5964-5971.