Phosphorylation of Cytokinin by Adenosine Kinase from Wheat Germ' Received for Publication July 20, 1976 and in Revised Form October 25, 1976

Home , Nucleoside, Phosphotransferase, Pyrophosphate

Plant Physiol. (1977) 59, 443-447

Phosphorylation of Cytokinin by Adenosine Kinase from Wheat Germ' Received for publication July 20, 1976 and in revised form October 25, 1976

CHONG-MAW CHEN AND RICHARD L. ECKERT Science Division, University of Wisconsin-Parkside, Kenosha, Wisconsin 53140

ABSTRACT monophosphates from the corresponding nucleosides using crude enzyme extracts of Acer pseudoplatanus. Laloue et al. (20) Adenosine kinase was partiafly purified from wheat genn. This en- reported the existence in vivo of cytokinin nucleoside-5-mono-, zyme preparation, which was devoid of adenine phosphoribosyltransfer- di-, and triphosphates in tobacco cells and A. pseudoplatanus ase and nearly free of adenosine deaminase but contained adenylate cells cultivated in liquid medium. Although these data, together kinase, rapidly phosphorylated adenosine and a cytokinin, N6_(A2_iso_ with the preceding evidence, demonstrated that adenosine ki- pentenyl)adenosine. Electrophoretic analysis indicated that only N6_(A2_ nase from plant sources could catalyze the conversion of cytoki- isopentenyl)adenosine-monophosphate was formed from the cytokinin nin nucleoside to its nucleoside-5'-monophosphate, the proper- while about 55% AMP, 45% ADP, and a trace of ATP were formed ties of this enzymic reaction still remained to be investigated. from adenosine. The biosynthesized nudeoside monophosphates were The kinetic study of cytokinin phosphorylation by adenosine quantitatively hydrolyzed to the corresponding nudeosides by 5'-nucleo- kinase in crude cell extracts is difficult because of interference by tidase and the isopentenyl side chain of the phosphorylated cytokinin other cytokinin-metabolizing enzymes such as adenosine deami- was not cleaved. The enzyme did not catalyze phosphorylation of ino- nase (adenosine aminohydrolase, EC 3.5.4.4) (4). sine. This paper describes the partial purification of adenosine ki- The phosphorylation of the cytokinin and adenosine required ATP nase from wheat germ cells and some of the properties of and Mg2+. The pH optimum was from 6.8 to 7.2 for both the cytokinin cytokinin phosphorylation. Phosphorylation of i6Ade and i6Ado and adenosine. At pH 7 and 37 C the Km and Vmax for the cytokinin by adenine phosphoribosyltransferase and adenosine kinase were 31 ,iM and 8.3 nmoles per mg protein per minute, and the values preparations from tobacco pith tissue cultures is also described. for adenosine were 8.7 jAm and 46 nmoles per mg protein per minute. Crude enzyme preparations from tobacco callus tissue and wheat germ MATERIALS AND METHODS phosphorylated N6_(A2_isopentenyl)adenosine. These preparations also phosphorylated N6-(A2_isopentenyl)adenine when 5-phosphorylribose-1- Chemicals and Enzymes. i6Ade, i6Ado, f-NADH, PRPP (so- pyrophosphate was present. dium salt), phosphoenolpyruvate, pyruvate kinase (rabbit muscle), lactic dehydrogenase (type I, rabbit muscle), adenylate kinase (rabbit muscle), adenosine deaminase (type I, calf intes- tinal mucosa), 5'-nucleotidase (Crotalus adamanteus venom), alkaline phosphatase (Escherichia coli), and wheat germ were obtained from Sigma Chemical Co. [8-'4C]Ado(50 mCi/mmol) The metabolism of cytokinins, N6-substituted adenine deriva- and [8-'4CJAde(50 mCi/mmol) were from Schwarz/Mann. The tives, in plant tissues has been extensively studied (4, 10, 13-18, preparation of [8-'4C]i6Ado(5 Ci/mol) and [8-'4C]i6Ade(5 Ci/ 25). The existence of enzyme systems which metabolize cytoki- mol) were as described (5). Tobacco pith tissue were grown on a nins in both plant and animal cells has also been well docu- medium as reported by Fox (11). The i6Ado-5'-P and its corre- mented (2, 3, 5, 6, 8, 9, 23). One of the major metabolites sponding diphosphate and triphosphate were a generous gift formed from natural or synthetic cytokinins is the corresponding from G. B. Chheda (Roswell Park Memorial Institute, Buffalo, nucleoside-5'-monophosphate. Fox and Chen (12) showed that N. Y). N6-benzylaminopurine is transformed into its related nucleotide Analytical Techniques. Phosphorylated compounds were ana- by cultured soybean callus. Others (13, 20, 25) also obtained N6- lyzed by paper electrophoresis (Camag TLE Cell, Whatman No. substituted adenosine-monophosphate as one of the cytokinin 3MM paper) with 0.5 M tris-citrate buffer (pH 3.5) at 20 C. metabolites. Electropherograms were cut into 0.5- or 1-cm sections and It was reported that mammalian adenosine kinase (ATP: placed in vials containing scintillation fluid (5). Radioactivity adenosine-5'-phosphotransferase, EC 2.7.1 .20) catalyzes the was measured in a Nuclear-Chicago Unilux II scintillation sys- phosphorylation of the cytokinin, i6Ado,2 and that the phospho- tem. Counting efficiency of paper chromatogram sections was rylated i6Ado is a competitive inhibitor of adenosine kinase, 74% for 14C. adenine phosphoribosyltransferase (AMP:pyrophosphate phos- A Cary model 14 spectrophotometer was used to measure the phoribosyltransferase, EC 2.4.2.7) and adenylate kinase rate of change in A. Protein concentration was determined by (ATP:AMP phosphotransferase, EC 2.7.4.3) (8). Doree and the method of Lowry et al. (22). Terrine (9) demonstrated the synthesis of N6-substituted-5'- Enzyme Assays. The adenosine kinase activity was measured either by the spectrophotometric method of Lindberg et al. (21) or by the paper electrophoretic method. The first method is ' This research was supported by National Science Foundation Grant as is BMS 72-02067AOI to C.-MC. based on the detection of a decrease in A at 340 nm NADH 2 Abbreviations: i6Ado: N6-(A2-isopentenyl)adenosine; i6Ade: N6-(A2- consumed in a coupled multienzyme system. The reaction mix- isopentenyl)adenine; i6Ado-5'-P, i6Ado-5'-DP and i6Ado-5'-TP: N6-(A2- ture contained in 1 ml, 20 gmol of P-enolpyruvate, 0.2 ,umol of isopentenyl)adenosine-5'-mono-, -di-, and -triphosphate, respectively; NADH, 0.5 ,umol of MgCl2, 50 ,tmol of KCI, 0.007 to 0.07 PRPP: 5-phosphorylribose-1-pyrophosphate. ,umol of Ado, pyruvate kinase (25 ,ug), lactate dehydrogenase (5 43 444 CHEN AND ECKERT Plant Physiol. Vol. 59, 1977 ,ug), and the enzyme preparation (15 ul). The reaction mixtures min, and the resulting supernatant was centrifuged for 2 hr at were always preincubated for 10 min at room temperature 80,800g. The supernatant, which contained the enzyme activity, (about 24 C) with all of the reagents before addition of Ado. was stable for several days when stored at -20 C. Since i6Ado was not a substrate for adenylate kinase, the spectrophotometric method could not be applied to this substrate. RESULTS The second method depended on direct measurements of radioactivity of reaction products after paper electrophoretic Partial Purification of Adenosine Kinase. The wheat germ separation of radioactive nucleoside substrates from nucleotides. extract was subjected to Sephadex G-100 column chromatogra- Unless otherwise specified, the reaction mixture (0.1 ml) con- phy. The protein was eluted with a solution of sodium phosphate tained 7 nmol of '4C-nucleoside, 0.4 ,umol of ATP, 0.3 Amol of buffer (0.004 M, pH 6.8), EDTA (0.001 M), and glycerol (5%). 5 MgCl2, ,umol of K-phosphate buffer (pH 7), and the enzyme Adenosine kinase activity was generally eluted between tubes 25 (5-60 ug protein). The reaction was started by adding the and 45 (5-ml tubes), with peak activity appearing at about tube enzyme to the reaction mixture which had been warmed to 37 C. 40 or about 0.5 bed volume (Fig. 1). The maximal peak area After incubation for 30 min at 37 C, the reaction was stopped by (tubes 35-43) was pooled and used in all assays. The degree of the addition of 1 volume of 95% ethanol. The mixture was then purification was approximately 20-fold when compared to the spotted on Whatman No. 3MM paper for electrophoresis. crude cell extract. This enzyme preparation had a specific activ- The assay of adenosine deaminase was as previously described ity of 19 to 25 units/mg of protein. It contained no significant (4). A Cary 14 spectrophotometer was used to measure the rate adenosine deaminase or adenine phosphoribosyltransferase ac- of shift in A from 260 nm (Ado) to 247 nm (inosine) or from 268 tivity. However, this enzyme preparation did contain adenylate nm (i6Ado) to 247 nm in 0.05 M phosphate buffer (pH 7.5) at kinase activity, as indicated by the presence of an ADP peak 24 C. The presence of phosphatase activity was measured by upon electrophoresis. Phosphatase activity which amounted to determining free inorganic phosphate according to King (19). about 5 to 7% of that of adenosine kinase was also present. No Adenine phosphoribosyltransferase activity was assayed by a inosine or Ado was formed from i6Ado (3), thus the isopentenyl modified radiochemical technique described by Thomas et al. side chain of i6Ado was not cleaved by this enzyme preparation. (28). Reaction mixtures contained in 0.3 ml of final volume: tris- Further fractionation of this enzyme preparation with ammo- HCI buffer (50 mM) at pH 7, MgCl2 (3 mM), PRPP (0.2 mM), nium sulfate did not yield any further purification but resulted in [8-'4C]Ade (0.02 mM), or [8-'4C]i6Ade (0.02 mM), and a crude a significant loss of enzyme activity. enzyme solution (2 mg protein). The reaction mixture was incu- Characteristics of the Enzyme Activity. The time course stud- bated for 1 min at 37 C before the addition of [8-'4C]Ade or [8- ies indicated that the rate of nucleoside phosphorylation reached '4C]i6Ade. The control tubes contained boiled enzyme or lacked a maximum in 20 to 30 min and then decreased (Fig. 2). The PRPP. After incubation for 30 min at 37 C, the reaction was decrease in enzyme activity after a 30-min incubation may be stopped by immersing the tubes in boiling water for 1 min. The due to the deterioration of the enzyme in aqueous solution and/ reaction mixture was then spotted on Whatman No. 3MM chro- or the limited presence of cofactors such as ATP. The linearity of matography paper with unlabeled AMP or i6Ado-5'-P as the Ado and i6Ado phosphorylation with respect to enzyme concen- marker. The mononucleotide product was separated from the tration is shown in Figure 3. Electrophoretic analysis of the substrate by electrophoresis. The electropherograms were cut reaction products after 30 min showed that only i6Ado-5'-P was and radioactivity was determined. formed from i6Ado, while about 55% AMP, 45% ADP, and a One unit of enzyme was defined as the amount that gave a V trace of ATP were formed from Ado. i6Ado was not a substrate value for phosphorylation of Ado of 1 nmol/min. The specific for adenylate kinase. activity was defined as enzyme units/mg of protein. The effect of pH on the reaction rate was determined at seven Extraction and Fractionation of Enzymes. The following steps pH values between 5.9 and 8 in 0.05 M sodium phosphate were performed at 2 to 4 C. Wheat germ (25 g) frozen with buffer. There was a broad pH optimum over the range 6.8 to 7.4 liquid N2 was homogenized in a Waring Blendor in 4 volumes/ for phosphorylation of both Ado and i"Ado. This is similar to the weight of 1 mm Na2EDTA. The homogenate was centrifuged for optimum range reported by Murray (24) for Ehrlich ascites- 10 min at 7,000g and the resulting supernatant was centrifuged for 2 hr at 40,000g. The supernatant was then dialyzed overnight against running1 mm Na2 EDTA. The dialysate was adjusted to pH 5 with 1 N acetic acid, and the resulting precipitate was I rapidly removed by centrifugation. The supernatant solution was adjusted to pH 6.8 with1 N KOH, and the precipitate was again 0 removed by centrifugation. The supernatant solution was lyophi- .l 4 lized, and the residue was dissolved in water in 0.1 of the volume ,E 4 EC' of the pH 6.8 supernatant material. This solution (6 ml, 378 mg S of protein) was layered onto a Sephadex G-100 column (2.5 x 0 e 84 cm) which had previously been equilibrated with a solution IL .2 containing phosphate buffer (4 mm, pH 6.8), EDTA (1 mM), and glycerol (5%). The protein was eluted with the same solution, and the fractions were analyzed for protein content and kinase activity. The fractions containing adenosine kinase were and further analyzed for adenosine deaminase, phosphatases, 10 20 30 40 so 60 70 80 adenine phosphoribosyltransferase activities. The enzyme prep- FRACTION NUMBER aration was stored at -70 C in the presence of 20% glycerol, FIG. 1. Purification of wheat germ adenosine kinase by Sephadex G- and the adenosine kinase was stable for at least5 months. 100 column filtration. Protein solution (6 ml, 378 mg of protein) was Crude cell extracts possessing adenosine kinase and adenine applied onto a column (2.5 x 84 cm) previously equilibrated with a phosphoribosyltransferase activity were prepared by homogeniz- solution of phosphate buffer (0.004M, pH 6.8), Na2EDTA (0.001M), ing wheat germ or tobacco pith callus tissue (35-day-old with5 and glycerol (5%). The protein was eluted with the same solution. volumes/weight of 0.05M phosphate buffer (pH 7.5) in a War- Fractions of 5 ml were collected and analyzed for protein and adenosine ing Blendor. The homogenate was centrifuged at 7,500g for 15 kinase activity. Plant Physiol. Vol. 59, 1977 CYTOKININ PHOSPHORYLATION 445 tumor cells with Ado as a substrate, but differs from the opti- Mg2+ concentration was 2 to 3 mm. The optimal ratio of ATP to mum of 6 reported for chick liver cells (23) when i6Ado was the Mg2+ was 1.3 to 2. At pH 7 and 37 C, the Km was calculated to substrate. be 8.7 ALM and 31 ,uM for Ado and i6Ado, whereas the Vm. was The phosphorylation of i6Ado and Ado was dependent upon 46 and 8.3 nmol/mg protein-min for Ado and i6Ado, respec- the presence of ATP and Mg2+. At 4 mm ATP, the optimum tively (Fig. 4). The Km for i6Ado is similar to the value (11.4 ,uM) obtained by Divekar and Hakala (7) using adenosine kinase from Sarcoma 180 cells. They also reported that i6Ado is a 8 competitive inhibitor of the enzyme (Ki 14.8 ,uM) when Ado is used as a substrate. Inosine was not a substrate for wheat germ adenosine kinase. Identification of Phosphorylated Metabolites. To determine if the nucleotide formed was a phosphorylated derivative of Ado 4 0 or i6Ado, the presumed monophosphate of the compound was eluted from the electropherogram. The eluate (one A2, unit of Ado or one A268 unit of i6Ado) was treated with 2 units (0.16 mg .2 protein) of alkaline phosphatase in 0.05 M tris-chloride buffer (pH 9.5) at 37 C for 2 hr. This treatment resulted in a loss of '4C-nucleotide and an increased level of '4C-nucleoside as demonstrated by paper electrophoretic analysis. Relative electrophoretic mobilities of nucleosides and nucleotides are shown in Table I. The evidence strongly suggested that the product was a phosphate ester, and it remained to be determined whether the 0 30 60 90 product was the 5'-monophosphate or the 3'-monophosphate. TIME (min) The presumed monophosphate of the compound eluted from the FIG. 2. Time course of cytokinin phosphorylation. The reaction mix- paper was digested with 1 unit (0.05 mg protein) of 5'-nucleotid- ture (0.1 ml) contained 7 nmol of 1[8_4CJi6Ado, 0.4 gmol of ATP, 0.3 ase of C. adamanteus venom in 0.05 tris-chloride buffer (pH 8.5) ,umol of MgCl2, 5 ,Smol of K-phosphate buffer (pH 7), and the enzyme at 37 C for 30 min. After this treatment (Fig. 5), the metabolite preparation (40 ,ug). The reaction was carried out at 37 C and the migrated with the corresponding unlabeled products nucleoside marker. were analyzed by paper electrophoresis. The experimental evidence indicated that the nucleotide was indeed the 5'-monophosphate. la Detection of Adenine Phosphonbosyltransferase Activity. 0 .2 0 0 The ability of wheat germ and tobacco callus tissue extracts to E E catalyze the phosphorylation of Ade and i6Ade by adenine phosphoribosyltransferase in the presence of PRPP as a phos- donor was .2 C phoribosyl also investigated before storing in freezer. 0S G-100 s Sephadex fractions was devoid of this enzyme activity. 41 However, a crude enzyme preparation prepared by high speed was able to the *0 centrifugation catalyze formation of AMP and i6 4 Ado-5'-P. Formation of nucleotides was not detected under the same experimental conditions in the absence of PRPP or with boiled enzyme. 0 10 20 30 40 50 60 0 10 20 30 40 50 60 PROTEIN (p) PROTEIN (lg) DISCUSSION FIG. 3. Effect of protein concentration on Ado and i6Ado phosphorylation. The conditions used were as described in Figure 2 except that The results of this show that protein study i6Ado may serve as a concentration was varied and the time of incubation was 30 min. substrate for the partially purified adenosine kinase from wheat

0.01o .E L

E 10.061. I o.o s EL0 jo.04 CP

0.02 KmS.7AN .1 i I 0 0.8 0." 0.24 032 i6uAdo, (M'xl1) [Ado]-! PA 4-4) FIG. 4. Reciprocal plot of adenosine kinase activity versus i6Ado and Ado concentrations. Experimental conditions were as described in except that i6Ado and Ado Figure 2, concentration was varied and the time of incubation was 30 min. 446 CHEN AND ECKERT Plant Physiol. Vol. 59, 1977

Table I. The Relative Electrophoretic Mobility of i6Ado and Some Related Nucleosides and Nucleotides Relative electrophoretic mobility refers to the mobility with respect to inosine. Electrophoresis was carried out on Whatman 3 MM paper in 0.05 M tris-citrate buffer, pH 3.5, for 3 hr at 240 v. Inosine Ade Ado i6Ade i6Ado AMP ADP ATP i6Ado-5'-P i6Ado-5'-DP i6Ado-5'-TP- Relative electrophoretic mobility -1.0 -8.0 -5.6 -6.0 -2.6 +1.4 +4.2 +6.2 +1.6 +4.8 +6.4

'i5A-do III Ade phosphoribosyl- i6Ade A transferase

x iSAdo-5-P i6AdoDP i6AdoTP 2 IA- +PRPP i 1 It wO .2 0 other .2 i6Ado-5'P 0 j U. I metabolites

x 0 51-nucleotidase 01 la (phosphatase) 5 0--B--o -5'-nucleotidase iljAdo 4 ... FIG. 6. Possible scheme for the interconversion of .q ~ A * +5 -nucleotidase cytokinin base and :1. 1 cytokinin-monophosphate. 3 Although the biological importance of cytokinin phosphoryla- .5li 2 tion is unknown, there are several pieces of evidence which allow us to speculate that the phosphorylation reaction may have a 1 0~~~I I 03 direct or indirect biological function. Scholtissek (27) reported 0 0 S.. ** that the phosphorylation of Ado by adenosine kinase appears to be related to Ado transport into chick fibroblast cells. A possible -20 -10 0 +10 +20 relationship between cytokinin STRIP NO. phosphorylation and transport remains to be investigated. Diveker et al. (8) reported that is FIG. 5. Characterization of phosphorylated cyytokinin by paper elec- Ado-5'-P, but not icAdo, was responsible for cytotoxic effects in trophoresis. A: Electrophoretic separation of phosphorylated i6Ado Sarcoma 180 cells. Furthermore, these authors demonstrated e: from its substrate i6Ado after incubation. For xperimental conditions that i6Ado-5'-P regulates the de novo synthesis of purine nucleo- see Figure 2. The amount of protein added was (60 gg, and the incuba- tides. Phosphorylated cytokinins may have similar tion time was 30 min. B: 5'-nucleotidase treatl ment of the presumed regulatory [14C]i6Ado-monophosphate. The''4C_labeled pez ak corresponding to j6 functions in plant cells. On the other hand, Hecht et al. (17), Ado-monophosphate on the electropherogram (iA) was cut and eluted. working with 9-deazaadenine derivatives and various other cyto- A portion of the eluate (about 2,000 cpm) was inc ubated with and kinin-active analogs, indicated that cytokinins can probably without ( ) 5'-nucleotidase. function without ribosylation, and that the free base itself might best fit the requirements of the "active" compound. The reports of incorporation of small quantities of cytokinin base into poly- germ, and iCAde may act as a substrate for thie adenine phospho- nucleotides (1, 12, 29) indicate that the cytokinin nucleotide is ribosyltransferase of wheat germ and tobalCCO with tissue ex- intermediate metabolite in the reaction. tracts. Since the enzyme preparation used is far from pure, these results offer no conclusive proof that phospl horylations of ieAdo Acknowledgment-We thank G. B. Chheda of Roswell Park Memorial Institute, Buffalo, N. and Ado are catalyzed by the same enzyyme. However, the Y. for providing i'Ado-5'-P, and its corresponding di- and triphosphate. finding that phosphorylations of Ado and ie Ado have the same pH optima (6.8-7.4) and ATP/Mg2+ ratio ( 1.3-2) suggests that LITERATURE CITED these two reactions are catalyzed by the seaime enzyme. If the 1. ARMSTRONG DJ, N MURAI, BJ TALLER, F SKOOG 1976 Incorporation of cytokinin N6- phosphorylation reactions are catalyzed by tthe same enzyme, it benzyladenine into tobacco callus transfer ribonucleic acid and ribosomal ribonucleic acid may be concluded that Ado has a higher affiinity for the enzyme preparations. Plant Physiol 57: 15-22 of the 2. CHEN CM, RL ECKERT 1976 Evidence for the biosynthesis of transfer RNA-free cytokinin. than ieAdo. This is indicated by the fact thai treplacemreplacementtofthe N6-amino group of Ado by an isopenteny/i amino side chain 3. CHEN CM, DM LOGAN, BD McLENNAN, RH HALL 1968 Studies of the metabolism of a decreases the Km value of the reaction by a factor of about 3.6. cytokinin, N6-(tA2-isopentenyl)adenosine. Plant Physiol 43: S-18. If the amino group is replaced by a hydr oxyl group to form 4. CHEN CM, 00 SMrrH, GF HARTNELL 1974 Biological activity of ribose-modified isopentenyl)adenosine derivative. Can J Biochem 52: 1154-1161 N6-(A2- inosine, this compound cannot act as a subs,trate for the kinase. 5. CHEN CM. OC SMIH, JD MCCHESNEY 1975 Biosynthesis and cytokinin activity of 8- Although it is still not clear whether or ne )t the cytokinin base hydroxy and 2,8-dihydroxy derivatives of zeatin and N'-(t-isopentenyl)adenosine. Bio- or its metabolite acts as the cytokinin in vivo, the preceding chemistry 14: 3088-3093 evidence together with other results (1, 9, 13, 20, 29) indicate 6. CHHEDA GB, A MITTELMAN 1972 N6-(A-isopentenyl)adenosine metabolism in man. Bic- chem Pharmacol 2t: 27-37 that there are enzyme systems in plant celIls that catalyze the 7. DIVEKAR AY, MT HAKALA 1971 Adenosine kinase of Sarcoma 180 cells. N6-substituted interconversion of cytokinin base to its coirresponding nucleo- adenosine as substrates and inhibitors. Mol Pharmacol 7: 663-673 tide. A possible scheme of this interrelation iship is presented in 8. DIVEKAR AY, HK SLOCUM, MT HAKALA 1974 N6-(A2-isopentenyl)adenosine-5'-mono- 2.72 wihcaay phosphate: formation and effect of purine metabolism in cellular and enzymatic systems. Figure 6. Adenosine nucleosidase (EC 3.2.. .7) which catalyzes Mol Pharmacol 10: 529-543 the irreversible hydrolysis of adenosine to adenine and ribose 9. DOREE M, C TERRINE 1973 Enzymatic synthesis ofribonucleoside-5'-phosphates from some has been isolated and purified from leaves c If spinach beet (26). N6-substituted adenosines. Phytochemistry 12: 1017-1023 The action of this enzyme from plant source on i6Ado remains to lo DYSON WH, JE Fox, JD MCCHESNEY 1972 Short term metabolism of urea and purine Plant 49: 506-513 )hate from ytokinin cytokinins. Physiol hatefrom Indoleacetic acid-kinetin be studied. Formation of cytokinin triphosp cytoksni 1. antagonism in certain tissue culture systems. Plant monophosphate (20) may be another branch in the metabolic Cell Physiol 5: 251-254 pathway. 12. Fox JE, CM CHEN 1967 Characterization of labeledribonucleic acid from tissue grown on Plant Physiol. Vol. 59, 1977 CYTOKININ PHOSPHORYLATION 447

'4C-containing cytokinins. J Biol Chem 242: 4490-4494 21. LINDBERG B, H KLENOW, K HANSEN 1967 Some properties of partially purified mammalian 13. Fox JE, J CORNETTE, G DELEUZE, W DYSON, C GIERSAK, P. Niu, J ZAPATA, J Mc- adenosine kinase. J Biol Chem 242: 350-356 CHESNEY 1973 The formation, isolation, and biological activity of a cytokinin 7-glucoside. 22. LOWRY OH, NJ ROSEBROUGH, AL FARI, RJ RANDALL 1951 Protein measurement with the Plant Physiol 52: 627-632 Folin phenol reagent. J Biol Chem 193: 265-275 14. Fox JE, W DYSON, D SOOD, JD MCCHESNEY 1972 Active forms of the cytokinins. In DJ 23. McLENNAN BD, A PATER 1973 The enzymic phosphorylation of N6-(A2-isopen- Carr, ed, Plant Growth Substances 1970. Springer-Verlag, Berlin pp 449-458 tenyl)adenosine in chick liver homogenates. Can J Biochem 51: 1341-1346 15. GUERN J, M DOREE, S SORDAGE 1969 Transport, metabolism and biological activity of some 24. MuutAY AW 1968 Some properties of adenosine kinase from Ehrlich ascites-tumor cells. cytokinins. In F Wightman and G Setterfield, eds, Biochemistry and Physiology of Plant Biochem J 106: 549-555 Growth Substances. Runge Press, Ottawa pp 1155-1167 25. PARKER CW, DS LETHAM 1973 Regulators of cell division in plant tissues. XVI. Metabo- 16. HALL RH 1970 Nl-(A2-isopentenyl)adenosine: chemical reactions, biosynthesis, metabolism of zeatin by radish cotyledons and hypocotyls. Planta 1 14: 199-21.8 lism and significance to the structure and function of tRNA. In: JN Davidson, WE Cohn, 26. POULTON JE, VS BuTT 1976 Partial purification and properties of adenosine nucleosidase eds, Progress in Nucleic Acid Research and Molecular Biology, Vol 10. Academic Press, from leaves of spinach beet (Beta vulgaris L.) Planta 131: 179-185 New York pp 57-86 27. ScHOLTISSEK C 1968 Studies on the uptake of nucleic acid precursors into cells in tissue 17. HECHT SM, RB FRYE, D WERNER, SD HAWRELAK, F SKOOG, RY SCHMrrZ 1975 On the cultures. Biochim Biophys Acta 158: 435-447 "activation" of cytokinins. J Biol Chem 250: 7343-7351 28. THOMAS CB, WJ ARNOLD, WN KELLEY 1973 Human adenine phosphoribosyltransferase. 18. KENDE H 1971 The cytokinins. Int Rev Cytol 31: 301-338 Purification subunit structure and substrate specificity. J Biol Chem 248: 2529-2535 19. KING EJ 1932 The colorimetric determination of phosphorus. Biochem J 26: 292-297 29. WALKER GC, NJ LEONARD, DJ ARMSTRONG, N MuRAI, F SKOOG 1974 The mode of 20. LALOUE M, C TERRINE, M GAWER 1974 Cytokinins: formation of the nucleoside-5'- incorporation of 6-benzylaminopurine into tobacco callus transfer ribonucleic acid. A triphosphate in tobacco and Acer cells. FEBS Lett 46: 45-49 double labeling determination. Plant Physiol 54: 737-743