474

BiochJraiea el B?ophysi*u Aclu. 1073(1991)474-480 © 1991 ElseviergeleneePublishersB.V. 0304M-165/91/$03.50 /IDONIS 0304416591001210

]~BAGEN23472

Catabolism of adenine nucleotides in suspension-cultured plant cells * N a r n i Y a b u l d a n d l-Iiroshi A s h i h a r a Department of Biology, Faculty of Science, Oehanoraizu Urriversity~ Tokyo (Japan)

(Re~ived 26 July 1990) Key words: Purinecatabnlisrn;Adeninenudeotide; Adenosine;Plant cell culture;(C roseus) Profiles of the eatabolism of adenine nneleotides in cultured pliant cells were investigated. Adenine nude-~,tides, prelabelled by incubation of suspension-cultured Catharantns ~osetts cells with |8-14C]adenosine, were catal~olized rapidly and most of the radioactivity appeared in 14COz. Allaatoin and allantoic aeid, intermediates of the oxidative catabolic pathway of purines, were temlmrarily labelled. When the cells, prelabelled with [8-MCladenoslne, were incubated with high concentrations of adenosine, the rate of catabolism of adenine nedeotides increased, a b e results suggest that the relative rate of catabolism of adenine nucleotides is strongly dependent oil the coneemrafion of adenine nuclcotides in the cells. Studies using alloperineL eoformyein and tiazofurin, inhibitors of enzymes Involved in purina metabolism, suggest the participation of AMP deaminase and xanthine oxidorednctase in the catabolism of adenine nucleotides in plant cells. AMP dealninase was ftmmd in extracts from C. rosens cells and its activity increased significantly in the presence of ATP. In contrast, no adenosine deaminase or adenine deaminase activity was detected. Qualitative differences in the catabolic activity of AMP were observed between suspension-cultured cells frem different speei~ of plants. Introduction In higher plants, there are several lines of evidence to suggest the presence of both the biosynthesis of adenine nueleotides de novo and their biosynthesis via salvage pathways, as shown in other organisms, such as mammals and batten'in ~1-5]. Most plants possess salvage enzymes, namely, adenine phosphodbosyltransferase and adenosine k i n a ~ [6,7]. Thus, their cells easily synthesize adenine nueleotides when adenine and adenosine are supplied exogenously. However, we are not in a position to conclude that the salvage pathways are actually functional within such cells because we have no data on whether or not adenine and adenosine are produced as the products of degradation of adenine nucleotides by the cells. Few studies have been carried out to investigate the catabolism of adenine nucleotides in plant cells. The aim of the present study was to ascertain the metabolic fate of adenine nucleofides in plant cells by * Part 38 of the scdes "Metabolicregulationin plant cell culture'. Correspondence: H. Ashihara, Department of BiOlogy,Faculty of ~cience~Ochanomizu Univelsity,2-1-1, Olsuka, Bplakyo-ku,Tokyo, 112, Japan.

tracing the flow of radioactivity from adenine nuclcotides, prclabelled by incubation of suspension.cultured plant celts with [g-tdC]adenosine, into various darienlives of purines. The results strongly suggest that adenine nucleotides are rapidly catabolized in plant cells. Metabolic routes and a mechanism for the regulation of catabolism of adenine uncleotides arc proposed from the results of several additional cxpertmenta. Materials and Methods Chemicals. Nutrients for culture media, allantoin, and uric acid were obtained from Wake Pure Chemical Industries, Osaka, Japan. [8-t4C]Adcnosine (spec. act. 1.84 G B q / m m o l ) was purchased from Amersham International, Ametsham, U.K. Coformy,~n was from Calbiochem, La Jolla, CA, U.S.A. Tiazofurin was supplied by the Drug Synthesis and Chemistry Branch, Division of Cancer Treatment, National Cancer Institute. Bethesda, MD, U.S.A~ All other chemicals were obtained from Sigma Chemicals, St. Louis, MO, U.S.A. Cell cultures. Suspension-cultured cells of Catharanthus raseus {L.) G. Don {= Vinca nTsea L), strain B, were maintained in 50 ml of Linsmeyer and Skoog medium [8] with 2,2 ~M 2,4-dichlorophenoxyacetic acid and 88 mM sucrose in 300-ml Erleumeyer flasks. The

475 conditions of culture were the same as those described in an earlier paper [9~. The growth of these cells can be divided into (i) a lag phase (day I)-ll, (ii) a logarithmic phase (day 1 7) and (iii) a stationary phase (day 7 - t 0 ) , Cells from each growth stage were used for the experimeats. In some experiments, suspension-cultured cells of betacyanin-producing Phytolacca americana [10]+ of Populus alba [11], a typical broad-leaved tree, and of Cryptomeria japonica ['12], an aeicular tree. were used. In these cases, cells from the cultures at the logarithmic phase of growth were used. Metabolism of [8-~+C]adenosine. Suspension-cultured cells (2O0 mg fresh weight) and 1.9 ml of culture medium from which the cells were isolated were placed in the main compartment of 30-ml Erlenmeyer flasks fined with a gJass tube that contained a piece of filter paper and 0.1 mt of 20~ K O H in a centre well. Unless otherwise indicated, reactions were started by the addition of 0.l ml of a solution of 100 ~M (8-~+C]adenosine (18.5 kBq) to the main compartment of the flask. The flasks were incubated in an oscillating water bath operated at 120 strokes/rain, with 5-cm amplitude, at 2 7 ° C for I h. After 1 h. the medium was removed a n d 1.9 ml of the culture medium and 0.1 ml of non-labelled 1 m M adenosine were added and the incubation was continued. After 1, 4. and 7 n, 20 pl of 6% perehloric acid were injected into the main compartment and the incubation continued for a further 5 rain, This acidification of the incubation medium resulted in the complete removal of 14CO2 from the medium. Absorbed potassium bicarbonate was elated with 10 t ~ of distilled water, a n d aliquots (1 ml) were used for the determination of radioactivity. Simultaneously, the ceils were harvested over Miracloth, washed wlth distilled water, and frozen in liquid nitrosen. Cellular metabolites a n d nucleic acids were extracted and analyzed as described in an earlier paper [13], with the single exception that 3% perchloric acid and 1 mM ATP in 80~ ethanol were used as the extraction medium in the pre~cnt stud2/. Determination of adenine nucleatides and their catabolites. Levels of ATP, A D P a n d A M P were estimated by three different methods, namely, spectrophotometry [141, luminometry [15], and analysis by H P L C [16]. Although essentially the same values were obtained after correction for recovery, relatively low recoveries were obtained when A M P and A D P were determined with a luciferin and luciferase system. Levels of purine nucleozides and bases were determined by HPLC using a column of poiyvinylalcohol gel, exactly as described in a previous paper [17]. Levels of allantoin a n d allamoic acid were estimated b y the method of Trijbeis and Vogels [181 with a slight modification, as described earlier [19]. Determination of the activity of deaminases. Extraction a n d determination of AMP, adenosine a n d adenine deaminases were performed essentially as described by

Yoshino and Murakami [21)]. The cclis (10 g fresh wt.) were homogenized in 0.1 M potassium phosphate buffer (pH 7.5) that contained 0.1 mM ditl~ othreitol and 0_4 M sucrose in a glass homogenizer. Tht. homogenate was centrifuged at 30000 x g for 5 rain a~ 2 ° C . The supernatant obtained was further centrifuged at 100000 × g for 21) nun at 2~'C. The supernatant was used for assays of the activity of various deaminases. For determination of the effect of ATP on the activity of AMP deaminase, the enzyme was partially purified by fractional[on with ammonium sulphate a n d ion-exchange chromatography of phosphocellulose as described by Yoshi~o and.[ Murakami [20]. The specific activity of A M P deaminase obtained from a typical experiment, under the optimal condition, was 240 amol substrate consumed per rain per mg protein. The preparation had no detectable activity of ATPases. The reaction mixture for the standard assay ,;onrained 30 mM cacodylate buffer (pH 7,1), 5 mM substrate+ 0.05,% bovine serum albumin and the preparation of enzyme. The amount o f ammonia liberated was determined by the method of Chancy and Marbach [211. Results and Discussion

Metabolic fate of adenine nucleotides in vivo In order to examine the metabolic fate of adenine nuclc0tides, suspension-cultured cells of Catharanthus roseus, obtained from three characteristic cultures at different stages o[ growth, were prelabefled with [8t~C]adenosine for I h, and then radioactivity was chased (Fig. 1). The preincubation method, using labelled adenosine or adenine, has often been used for studies of the catabolism of adenine nucleotides in mammalian cells [22-24]. After incubation of Catharanthm cells with 5 t i m [8-~4C]adcnosine for 1 h, more than 85~ of the radioactivity was distributed in the salvage products, i.e., nucleotides a n d nucleic acids, The predominant labelled products were ATP and RNA, and only small amounts of guanine nueleofides were labelled (data not shown). When the prelabelled cells were incubated with non-labelled 50/~M adenosine, 154--75% of prelabelled nucleotldes disappeared within 1 h, and~ concomitantly, a large amount of radioactivity was di+~ tected as C O z. It is noteworthy that 60-75% of the total radioactivity taken up by the cells was converted to t'tCO2 during the chase. The release of 1'*CO2 from |8-14C]adcnosine was almost completely inhibited when the cells were incubated with 0.5 m M allopurinol, an inhibitor of xanthine oxidoreductas¢ [251, and the radioactivity was concomitantly accumulated in xanthine (Table I). Xanthine oxidoreductase catalyses the conversion of hypoxanthine to xanthine as well as the conversion of xanthine to uric acid. In our culture system, only xanthinc, and not hypoxantkine, was accumulated in the cells as a result of the treatment with 0.5 mM

476

with t~s hypothesis, the rate of incorporation of radioactivity from [8-ZaC]adenosine into the salvage products was found to be slightly increased in the altopurinottreated cells when the rate was expressed as in terms of percentage of total uptake (Table I). Accumulation of xanthine as a result of treatment with allopurinol has been reported in seedlings of leguminous plants [19,2.8]. By contrast, treatment with allopurinol caused accumulation of hypoxanthine in cultured soybean cells grown with IMP as the sole source of nitrogen [29]. Results of pulse-chase experiments also showed ternporary increases in the radioactivity in allantoin and allantoic acid (Fig. 1). These results strongly suggest that adenine nucleotides are calabolised by the conventional oxidative catabolic pathway of purines, wh/ch ts also widely distributed in animals and microbial organisms 13]. Although rapid synthesis and degradation of adenine nueleotides were observed in Cathaeanthus ceils at every stage of growth examined, the rate of catabolism of adenine nucleotides increased slightly with the age of the ceils, However, the rate of degradation of nucleic acids was rather lower in the cells in the lag phase than in the cells at other phases of growth. The higher rate of degradation observed in cells at the logarithmic phase

TABLE I Effects of allupurinol on .~hemetabohsm of {8- I~¢Z]adenosme by sus.ven. yion oultured Catharanthus roseuy cells Four-day-old cells ~ueee incubated with 5 /~M [8-Sac]adenosine and 0,5 ,a~M allopurinol for 6 h. The average haloes and S D . were obtained from dupli:at¢ samples in a typical experiment. The rates of incorporation of radioactivity are expressed as kBq g fresh kvl. - I and as ~o of total uptake (in parentheses). Mat abolitc~

Incorporation (kBq g fresh wt. ~) allopurinol (0.5 haM) A (~) c

control,

Salvageptoducts" 16.8+0.8 (78.5) 15.4+0.1 (82.4) Xanthin¢ 0.2~0.t (0.9) 9:1:L0.1 (14AS Catabolites ~ 4.04-0,4 (18.7] 0.34-0.0 (1.6l Others 0.4-1-0.1 (1.9) 0.3.t:0.1 (1.6) Total 21.4 + 1,4 (tOO) 18.7.L0,2 (100)

+3.9 +13.5 -I7,1 -0.3

Salvageproductsconsistof nucleicacids andanti.tides. h Camboliles consist or allantoin, allaatoic acid and. CO 2. c A indicates the changes in the relative lb~vel(~) or a metabolite as a result of the p~slmc¢ of allopurinol.

allopurinol. This result may be due to the fact that no xauthine, but only hypoxanthine, is salvaged by the reaction catalyzed by hypoxanthine-guanine phosphoribosyltrartsferase 126,27]. Thus, no accumulation of kypoxanthine would be observed in the cells. Consistent

m

-~ BO

)

m~

)

W

a

i

,Nuc.

012

C

It/e

"/if o

I

b

5

i

80 ] Time

2

5 of incub~tt i o c

80

2

5

8

(hr)

Fig. L Distribution in pulse-c]~ag~ ~xpefiments of tsd~0activiw from [S-14C]aden0~in~ in trleIabolit©s of suspcnsion-,.'altu~;d c~tls of Catlmwa~lhw~ rosens at different stages of growth: a, lag phase (day 1); b, legadtlm-dc phase (day 4); c. stationary phase (day 10). The c=lLs ,.here incubated with Linsm~yer and Skoog medium that contained 5 ~M tS-:Cladeaosine for 1 h (pulse) and then the incubation medium was replaced by medium that contained 50 ~tM non-hballed adenosine, Incorporation of the radioactivity is eaprcssed as a percentage of total radioactivity. The total radioactivity taken up by the 1-, 4- axed 10-day-old cells ,xas 13.2±2,6, 15.1+1.7 and 14.3+3.2 k B q / g fresh wt.. respectively. The values were averages from duplicate samples in a typical experiment. 0, Nuelentidcs; o, nucleic adds; za, allaat0in; a.. alianloic acid; lit C02; D,

adezune+ adenosine,

477 m a y reflect a rapid rate of turnover of nucleic acid~ at this stage. The degradation of nucleic acids was also rapid in the stationary phase, when net degradation of nucleic acids was also observed [27 I. lntracellular levels of adenine nucleotides and their catabolites The results of pulse-chase experiments strongly suggest that adenine nucleotides were rapidly catabolised and purine rings were immediately eleavaged in Catharanthus cells. If these events occur similarly in cells in vivo, only small amounts of the intermediates invotved in the catabolism of purmes can be accumulated in the cells. To confirm this possibility, we determined intraeellular levels of adenine nucleotides and their catabolites. C o m p a r e d with levels of adenine nucleotides, the levels of purine nucleos~des, bases and ureides were extremely low and most of these compounds were not detectahle in the extracts of the cells (Table I1). Although adenosine was always found, its levels were only 0.8-4.1% of the total level of adenylates. Low levels of adenosine have also been reported in suspension-cultured Datura mnoxia cells ( 0 . 2 - l . 5 n m o l / g fresh wt.) [31] and in Nicotiana tabacum cells (0.8-3,7 n m o l / g fresh wt.) [32], A small a m o u n t of adenine was found only in cells in the logarithmic phase (Table II). It has been proposed that enzymes involved in the catabolism o t p u d n e s are distributed in the eytosoL the pernxisomes and the endoplasmie retienlum [32]. Neverthdess, the present results indicate that

TABLE n LeuetJ of Lzdenme nucleoledes and lhe~r cot~bolffe,~ m su.~enyi~n~ultured Catharanlhm roseu.¢cells Levels of adenine nu¢lcotides, adenosine and adenine are expressed ag nraol/g rr~h weight The average ,,minesand S.D. were obtained from doplicate samples using differem extracts in a typical experiment. Total adenylale (AN). adenylate energy charge, i.e.. [ATP] + ~[ADP]/IANL and the ralio, (adenosine4-adenine)/AN ate also shown.

ATP" ADP" AMP= Adenosine (AR) b

Levels (nmol/g (~esh wt,} day I day 4 day tO 222.3±59.2 203.2-4-20.8 117.4±5.2 26.7±21.6 42.g± 0.4 3IA-~ 5.5 35.2± 3.1 35.6±213 21.8± LS 9.] :~ 1.3 2.2 ± 1,2 7.O + L2

Adenlne (A) b

n,d.

AN

285.2

281,6

0.83 0.03

0,~) 0.01

Energy CharRe [A + A R I / A N

0,3± 0 2

n.d.

]70.6 0.7S 0,04

The following compounds were undetectab]e in extralets Of cultures at

any stage: inosine~ hypoxanlhine, xanthlne, urlc acid, al|anloin anA al]antoi~; a~d. a Determined by Sl~Ctmphotom©lrica~say [14]. h Determined by HPLC [17]. n.d., not detectable.

60

~0

Nuc[ercacids

~ 20

0

~--

125

t

r

12.5

125

Adenosine

(pM}

Fig. 2. Effects of Ihe cor,centration of adano~ianon the metabolic fate of adenine nucl~,odd~sp~lahellcd with [8:aCladenosine. Cazb.aranms rosent~ cell~ (day 4) were incubated in medium thai c~_talrled 5 ~M lS:4Cladenosine for 1 h, and then the incubation medium was replaced by fresh medium that conlaincd various concentrations of adenosine, Incorporation of radic~activityis ~pt-~.scd a s pcrr:,cntage of total uptake. The average values and S_D. were obtained from duplicate samples in a typical experiment. Averaged values and SD. of total radioacti',ity taken up by the ¢~lls oblain=d from six samples were 17.2 ±1.0 kBcL/g fresh ~ . o. Nuclcobd~; o , nuel~c acid; at. CO.,; a, uTeides(i.~, allant~[n plus allantoic acid).

catabolism of ureides is very rapid in Catharanthus ceils. This observation suggests the presence of channeling of the catabolic pathway. T h e levels of A T P were always m u c h higher than those of A D P a n d A M P . T h e values of the adenylale energy charge in l-, 4-, and 10-day-old ceils were almost constant. T h e rapid degradation of A M P m a y participate in the control of the adenylate energy charge in the cells, as ha~ b ~ suggested to be the case m other organisms [34,35]. Effects o f the concentration o f adenosine on the metabolic fate o f adenine nudeotides in order to examine whether intracellular levels of adenylates influence the metabolic fate of adenine nucleotides, suspension-cultured Catharanthus roseus cells were prelabcUed with 5 p.M [8-14C]adenosine for 1 h, as described above, a n d the radioactivity was chased by a n l a b d l e d adenosine at three different concentrations. After 3 h, distribution of the radioactivity was determined (Fig. 2). With increases in the concentration of the unlabelled admaoslne, the distribution of 14C to the catabofites, i.e., to ureldes and C O 2, increased dramatically. C o n c o m i t a n t l y , r a d i o a c t M t y in the nucleotide and nucleic acid fractions was rapidly reduced. The I c v d of A T P was changed by the exose-

TABLE Ill Effect~ oJ the t~n¢~ntration ef adenosine on 1he [e~,elof ATP in suspension.cultured Cotharenthus rosew cel(s Four-day-old cells (3~0 nag fresh wt.) were incahated for 3 h with 3 ml of fresh culture medium that contained different concentrations of adenosine. The level of ATP was determined lum]nometricaily [15]. The average values and S.D. were obtained from duplicate samples using different extracts in a typical experiment. Adenosine

Level of ATP (nmol/g fresh wh)

O,O 1.25 12.5 12.~

13%2i 6,5 183.7 + 18.5 152.1 ±34.8 300.0-- 0. t

(%) (l(R)) 1134) (111) 1219)

TABLE IV Effects of coformycin and tiazo[urine on the metabolism of [8. I¢CJadenosine by au.spemiorl-cultured Catharanthua rosegs ceils Four-day-old cells were incubated for 1 h with 5 pM [8-mC|aden~ine and chased for 3 h with 125/~M non-labelled adenosine. "l'he average values were obtained frrwn duplicate samples in a typical experiment. The rates of uptake of radioactivity are expressed as kBq/g fresh weight and incorporation of radioactivity into cellular components is expressed as a percentage of total uptake. Values in parentheses indicate the change in the proportion (%) of the total radioaeti,hty in a given metabolites that resulted from the presence of an inhibitor. Metahalites

lncorporatlon ({g of total uptake) control

nously supplied adenosine, but a significant increase in the level was detected only at the highest concentration (125 ~ M ) of adenosine tested (Table Ill). T h e s e results support the hypothesis that intracellular levels of adenine nucleotides are controlled through the d e g r a d a tion of the adenylates. Possible pathways o f catabolism o/adenine nuclear/des T h e catabolism o f adenine nuclear/des is initiated b y d e a m i n a t i o n and dephosphorylation reactions. Several p a t h w a y s have been proposed for different organs in m a m m a l s [3]. Theoretically, the presence, in plant cells, of the following p a t h w a y s can be proposed: AMP ~ IMP -÷ inns/no ~ hypoxaathlne

(1)

AMP ~ IMP ~ XMP ~ santhosine ~ ~anthine

(2)

AMP ~ adenosine ~ inns/no ~ hypoxanthine

(3)

AMP ~ adenosine ~ adenine ~ hypoaanthine

(4)

T o clarify the major p a t h w a y of catabolism of A M P in viva, we first e x a m i n e d the effects of coformyein, an inhibitor of adenosine d e a m i n a s e a n d A M P d e a m i n a s e [36], and the effects of tiazofurin, an inhibitor o f I M P dehydrogenase [37] on the m e t a b o l i s m of adenosine {Table IV). Coformycin inhibited the catabolism of purines and, as a result, A M P a n d not adenosine was accumulated in the cells. In contrast, little or no effect of tiazofurin on the m e t a b o l i s m of adenosine was observed (Table IV). These results eliminate p a t h w a y s 2 and 3, Pathway 1 seems the m o s t plausible. Consistently, we confirmed the presence of A M P deaminase in crude extracts f r o m Catharamus roseus cells. Significant activity was only found in the presence of A T P (Table V), Stimulative effect of A T P o n the activity of A M P d e a m i n a s e was also c o n f i r m e d in the partially purified preparation (Fig. 3). Thus, catabolism of A M P seems to he strongly influenced b y the conceatration of A T P in the cells. Activation of this enzyme hy A T P has been found in other p l a n t species

Salvageproduetsa 15.1 AMP 1_4 Catabolites b 82.2 Adenosine 0.01 Othar~ 2.7 Total uptake (kBq/g)

coformycin (5 ~M) ti~ofm'in (100 pM) 40.0 (+24.9) 5.0 (4-3.6) 57.8 {-24.4) O.03 (+0.02) 2.2 (-0.5)

56.9 :h 0.8 zl~..g± 4.6

].6.7 (+1.6) 0.6 (-0.8) 81.9 (-0.3} 0.021+O.01) lA (-1.3) 49.7. + 0.8

Salvage products consist of nucleic acids and nuclear/des including AMP. i, Catnboliles consist of a~lant0Jn, allantoic acid and C02. [20,38]. N o significant activity of adenine d e a m i n a s e a n d adenosine d e a m i n a s ¢ was found in the cells (Table V)Urn/des, namely, allantoin a n d allantoie acid, seem to be f o r m e d f r o m h y p o x a n t h i n e b y w a y o f xanthine and uric acid. W e did n o t e x a m i n e the route o f d e g r a d a tion o f urn/des in the present study. Nevertheless. the release o f large a m o u n t s o f t~CO: f r o m [8-t4C]AMP, prelabelled with [8-t4C]adenosine, indicates that s o m e of the purine rings were converted to C O a a n d probably to N H 3 in the cells. Possible catabolic p a t h w a y s of urn/des have been reviewed by T h o m a s a n d Schrader [39l, Metabolism o f [8-J'C]adenusine in suspension-cultured eell,v o f other planS species Pulse-chase e x p e r i m e n t s were carried o u t u s i n g three further types o f suspension-cultured cell, In addition to TABLE V Activity of deamlnages involved m catabel~sra of adenine nuclear/des (n extracts frost! saf.oension-eultured CatharanZhas roaeus cells Cells at the stationary, phase (day tO) were u~ed, Enzymatic activities are expressed as nmol/min per mg protein. The values and SD. were obtained more than four different preparations of eratyme. Subslrale AMP (S raM) AMP (S raM) Adoao~irm (5 mM) Adenine (5 mM) n.d., not detected.

Addition

A'FP (5 raM)

Activity (nmol/mln per mg protein) 0.28 +0.03 2.8 +0.6 n.d, n,d.

479

250 •

~ 200 Q_

"It

.

A TP

ArP2

5 mMw

5r~M

j~.l-

E c

150

z

100

• ~..J

-Arp

1

2 3 4 5 [AMPI (raM) Fig. 3, Effect of AMP concentration on the acti~qtyof AMP deaminase partially purified from smpension-euhured Calharanthus roseus cells. The a~ti,dty was assayed in the presence or absence of A'I'P. "The values were obtained [rem a typical experiment, n, Minus ATP: &, with 2 mM ATP. D, with 5 rnM ATE

the salvage pathway for adenosine, the catabolic pathway of adenine nucleotides also functions in these various cells (Table VI). However, the patterns o f degradation of adenine nueleotides were somewhat different

Acknowledgements W e thank Dr. H. Sasamoto, Foregl and Forest Product Research Institute. Tsukuba, Japan, and Dr. M. Sakuta o f our D e p a r t m e n t for their generous supply of cultured plant cells. W e are also indebted to the National Cancer Institute, Bethesda, U.S.A, for a suppl 7 of tiazofurin. This research was supported by a Grant-inA i d for Scientific Research (No. 01540560) from the Ministry of Education, Science a n d Culture, Japan.

References

TABLE VI Metabolism of [8-J~C]adenoslne in ~t~spension-cultured cells of pokew@@d(Phytolacca americana), poplar f Populus alba) and Japanese cedur tCo'ptomeria japanifa)

The cells were incubated for 1 h with 5 /~M [gJaC]adenosine and chastd for 3 h with 50 pM non-labelled adenosine, Cells at logarithtalc phase were used for experimmtts. The incorporation of radioactivity is expre~ed as a percentage of told up~kc. The total radioactivity mken np by the cells is expre~ed as kBq./g fresh w¢i~t. Fraction

one from another. A very low rate of catabolism of adenine nucleotides was found in pokeweed cells. In poplar cells, ureides were produced from AMP, but the rate of further degradation of ureides was extremely low. Metabolism in cedar cells resembled that in Catharanthus celts, b a t the m e t a b o l i s m of [8tOC]adenosine taken up by the cells was very slow. The radioactivity i n the fraction of adenine plus adenosine fraction, which mostly consisted of unmetabotized adenosine, accounted for 68% of the total radioactivity during the period o f the pulse (1 h) and approx. 65% of if'As radioactivity was transferred 1o the other comp o u n d s ia the course of 3-h chase. Tke differences in the patterns of purina catabolism a m o n g these cultured cells may reflect the metabolic capacity o f the original tissues. Differences in the metabolic fates of [8m~Cladenine and [8-taC]hypoxanthine in several plant tissues have been reported [13.40].

Incorporation {% of total uptakel (a) pokeweed

(b) poplar

(c} cedar

Full= {I h) Nueleotides 50.2¢-7.'/ Nucleic acids 45.04- 8.6 Ureid©s 1,0~ 0,0 CO z 0.2+0.0 Adenia©÷ adenosine 1.74-1.5 Others 2,0±0,5

47.1 4-7,0 31.24- 5.4 13.3 + 1,4 0.4+0.0 5.94-1.2 2.1 4- l,I

14,1 -I-0.8 I 1,3 + 1,2 0A + 0.0 3.3.t:0.1 67.8 4-2.5 33 t-0.6

Cha~ (3 hi Nuctcolides Nucleic acids Ureides COz Adenine+adenosine others

37.8=[:0.5 ~2.3± L0 5.0± 0.3 1.520.1 1_9J. 0,2 1.5.4-0.1

38.5 ±0.4 22.7± L3 37.0±0,7 0.6±0.0 1.2±0,2 0.04-0.1}

25.0±2.7 20.8 ± 1.2 IA±0.t 20.7 4-1.5 23,84-4-9 8.6+0.2.

Total uptake (kBq/,@

15.65:2.4

~0.3 ±8.9

5.0±0.2.

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Catabolism of adenine nucleotides in suspension-cultured plant cells.

Profiles of the catabolism of adenine nucleotides in cultured plant cells were investigated. Adenine nucleotides, prelabelled by incubation of suspens...
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