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Metabolism of cytokinin: Phosphoribosylation of cytokinin bases by adenine phosphoribosyltransferase from wheat germ
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 214, No. 2, April 1, pp. 634-641, 1982
Metabolism of Cytokinin: Phosphoribosylation of Cytokinin Bases by Adenine Phosphoribosyltransferase from Wheat Germ’ CHONG-MAW CHEN,’ DEBRA K. MELITZ, AND FRED W. CLOUGH Department
of Life Science and Biomedical Research Institute, University Kenosha, Wisconsin 53161 Received July 15, 1981, and in revised form November
of Wisconsin-Pa&side, 13, 1981
Adenine phosphoribosyltransferase (AMP:pyrophosphate phosphoribosyltransferase EC 2.4.2.8) which catalyzes the phosphoribosylation of cytokinin bases and adenine to form the corresponding nucleotides were partially purified from the cytosol of wheat (Triticum aestivum) germ. This enzyme (molecular weight, 23,000 + 500) phosphoribosylates the bases at an optimum Mg2+ concentration of 5 MM and optimum pH of 7.5 (50 mM Tris-HCl buffer). K, values for NG-(A’-isopentenyl)adenine, NG-furfuryladenine, NG-benzyladenine, and adenine are 130, 110, 154, and 74 PM, respectively, in 50 mM Tris-HCl buffer (pH 7.5) at 37°C. Hypoxanthine and guanine are not substrates for the enzyme. In concerting with other cytokinin metabolic enzymes, this enzyme may play a significant role in maintaining the supply of adequate levels of “active cytokinin.”
Cytokinins are a group of plant hormones which regulate cell division and differentiation (l-3). Cytokinin bases and cytokinin ribonucleotides have been found in various plant cells, and these cytokinins are metabolized in the plant cell to form different metabolites (4-11). The relative amount of a specific metabolite varies in different tissues under different physiological conditions. Doree and Guern (12) studied short-term metabolism of some exogenous cytokinins in Acer peudoplatanus cells and observed that one of the major metabolites formed from cytokinin base was the corresponding nucleotide. Their observation suggests that in plant cells there is an enzyme system catalyzing the formation of cytokinin ribonucleotides from their corresponding bases. Although
phosphoribosylation of Ade to form AMP by adenine phosphoribosyltransferase has been widely studied in animal (13-15) and microbial (16-18) cells, there have been very few studies of this enzyme from plant cells; furthermore, the role of this enzyme in cytokinin metabolism in plant cells has not been defined. We report here the partial purification of adenine phosphoribosyltransferase from wheat germ, the properties of the enzyme, and the kinetics of the phosphoribosylation of cytokinin bases by this enzyme. MATERIALS
Chemicals and Enzymes Ade, AMP, i6Ade,3 NG-benzyladenine, NG-furfuryladenine, or kinetin, other ribonucleotides, Carbowax (Mr wt., 6000), 5’-nucleotidase (crotalus adaman-
1 This work was supported by the National Science Foundation Research Grant PCM 79 03832 to C.-M.C. ZAuthor to whom all correspondence should be addressed. 0003-9861/82/040634-08$02.00/O Copyright All rights
0 1982 by Academic Press, Inc. of reproduction in any form reserved.
AND METHODS
3 Abbreviations used: PRPP: 5-phosphoribosyl-lpyrophosphate; i’Ade: fl-(A*-isopentenyl)adenine; i’Ado: fl-(A’-isopentenyl)adenosine; P, phosphate. 634
CYTOKININ BASE PHOSPHORIBOSYLATION teus venom), 5-phosphoribosyl-1-pyrophosphate, and wheat (Triticum aestivum) germ were from Sigma Chemical Company; i’Ado-5’-P was from P-L. Biochemical Company; [8-‘4C]hypoxanthine (62 &i/p mol), N6[8-i4C]furfuryladenine (15 &i/pmol), and N6[8-i4C]benzyladenine (13.4 &i/pmol) were from Amersham Corporation.
Analytical
Methods
Protein concentration was determined according to the method of Bradford (19) using bovine serum albumin as a standard. A Cary Model 14 spectrophotometer was used to quantify purine bases and nucleotides. Nucleotides, nucleosides, and bases were separated by paper electrophoresis (Camag TLE Cell, Whatman No. 3MM paper) with 0.1 M Tris-citrate buffer (pH 3.5) at 20°C and/or paper chromatography (Whatman No. 3MM) in a descending fashion using the following solvent system (v/v): A, ethylacetate:l-propanol:HzO (4:1:2), B, 1-butanol: HzO:concentrated NH,OH (86:14:5); C, 2-propanol: HzO:concentrated NH,OH (721); D, 95% ethano1:O.l M (NH&B03 (pH 9.0) (1:9), E, 1-propanol:concentrated NHIOH:HzO (60:20:20).Sephadex LH-20 resin (Pharmacia) was swollen in 35% aqueous ethanol and the column was eluted with this solvent. Radioactivity was measured in a Tracer Analytic Mark III liquid scintillation system. For liquid samples, an aliquot of no more than 0.5 ml was added to 10 ml of Bray’s solution (20). For paper chromatograms, l- or 2-cm sections were placed in vials containing scintillation fluid (21).
Preparation
of [8’4C]i6Ade
[8-‘4C]i6Ade was synthesized from 3-methy -2-butenylamine (hydrochloride) and 6-[8-‘4C]chloropurine (22). The former compound was prepared according to the procedure of Hecht et al. (23). 6-[B‘*C]Chloropurine, commercially not available, was synthesized from [S-“Clhypoxanthine by the method of Bendich et al. (24). The synthesized [8-‘*C]i6Ade was purified by Sephadex LH-20 column and then by paper chromatography in solvent system C. The synthesized radioactive i’Ade had uv absorption spectra Amax272 nm at pH 2; 269 nm at pH 7, and 275 nm at pH 12. Paper chromatographic mobility of [8-14C]i6Ade was corresponded to that of authentic i’Ade in five solvent systems (A to E).
Extraction
and Fractionation
of Enzyme
Wheat germ (200 g) frozen with liquid N2 was homogenized in a Waring Blendor in 4 vol/weight of buffer A (50 mM Tris-HCl, pH 7.5, 5 mM MgCla, and 1 mM mercaptoethanol). The homogenate was centrifuged for 10 min at 15,000~ and resulting super-
635
natant was centrifuged again for 30 min at 20,OOOg. The supernatant is referred to as crude extract. The following steps were employed to purify the extract further. All procedures were conducted at 0 to 4°C. AMP and (NH&SO4 were Step 1: Heat treatment. added to the crude extract to a final concentration of 1 and 30 mM, respectively, and the mixture was heated for 5 min in water bath at 60°C. After cooling to 4°C in an ice bath, it was centrifuged for 30 min at 20,OOOgand the precipitate was discarded. Step 2: Ammonium sulfate fractionation. Solid ammonium sulfate was added to the collected supernatant to make 30% saturation. After 1 h the sample was centrifuged at 20,OOOgfor 20 min. The 30% supernatant was brought to 80% saturation by addition of solid ammonium sulfate, and the precipitate was again collected by centrifugation at 20,OOOgfor 20 min. The pellet was resuspended in buffer A and dialyzed for 18 h against buffer A. The dialysate was reduced to 8 ml by Carbowax. Step 3: DEAE-cellulose chromatography. The concentrated protein solution (207 mg protein) was applied onto a DEAE-cellulose (Whatman DE-23) column (2.5 X 28 cm) equilibrated with buffer A. The column was eluted with 0.72 bed vol of this buffer, followed by the same bed volumes of 0.25 M KC1 in buffer A, and finally eluted with 0.5 M KC1 in this buffer. The adenine phosphoribosyltransferase fractions were pooled and dialyzed against buffer A for 17 h. The protein solution was reduced to 7 ml by Carbowax. Step 4: Sephadex G-75$ltration. The enzyme solution (28 mg protein) was filtered through a Sephadex G-75 column (2.5 X 31 cm) and eluted with buffer A. The fractions containing adenine phosphoribosyltransferase activity were pooled and reduced to 5 ml. Step 5: Sephadex G-.%Nchnnnatography. The Sephadex G-75 fraction (5.6 mg protein) was further chromatographed on Sephadex G-200 column (2.5 X 35 cm) equilibrated with buffer A. The enzyme was eluted with the same buffer and the active enzyme fractions were pooled, reduced to 10 ml by Carbowax, and stored at -20°C.
Enzyme Assays The adenine phosphoribosyltransferase activity was measured either by paper chromatographic method or by butanol extraction method. The first method depended on direct measurement of radioactivity of reaction product after chromatographic separation. The second method is based on the differential solubility of substrate and reaction product. In the first assay method, the reaction mixture (170 ~1) contained 36 PM [8-14C]Ade,or 32 pM [8-i4C]i6Ade, 5 mM MgClz, 1 mM fl-mercaptoethanol, 600 pM PRPP, 50 mM Tris-HCl buffer (pH 7.5), and the enzyme (1560 pg protein). The reaction was started by adding
636
CHEN, MELITZ, AND CLOUGH TABLE I PURIFICATION OF WHEAT GERM ADENINE PHOSPHORIBOSYL TRANSFERASE’
Fraction
Total protein (mg)
1. 20,000g supernatant 2. Heat treatment 3. 80% (NH&SO1 4. DEAE-cellulose pool 5. Sephadex G-75 pool 6. Sephadex G-200 pool
4955 902 207 28 5.6 2.4
Specific activity (nmol/min/mg) 0.2 1.0 3.3 11.9 33.5 41.3
Total activity (nmol/min) 991 902 683 333 188 99
Recovery (%I 100 91 69 34 19 10
Purification (n-fold) (1.0) 5 17 60 168 207
a The starting material was 200 g wheat germ cells. Enzyme activity was determined as described in the text with Ade as a substrate. The paper chromatographic assay system was used. the enzyme to the reaction mixture which had been warmed at 37°C. After incubation for 15 min at 37”C, the reaction was stopped by the addition of 100 11 of 95% ethanol. The mixture was spotted on Whatman No. 3MM paper with AMP or i’Ado-5’-P as carrier, and the chromatogram was developed with solvent D. In the second assay method, the reaction mixture containing components identical to the first method was incubated for 15 min at 3’7°C. At the end of the reaction, 2 ml of water-saturated butanol was added to the reaction mixture and the content was mixed thoroughly. The upper butanol phase was carefully siphoned from the lower water phase. This butanol extraction procedure was repeated eight times. About 90% of Ade was found in the butanol phase. The water phase containing [8-14C]AMP and less than 10% of Ade was transferred to scintillation vial and the radioactivity was determined. The background counts (10%) were substrated from the total counts. This butanol extraction method was used only in locating the enzyme fractions eluted from column chromatographies. Furthermore, since cytokinin nucleotide is soluble both in butanol and water, this method was not employed for cytokinin nucleotide synthesis assay. RESULTS
Pur$icatim of Adenine Phosphtibosyltransferase
Purification of this enzyme from 200 g of wheat germ cells is summarized in Table I. In the heat treatment step, 30 mM (NH&SO4 and 1 mM AMP were added to the crude enzyme extract to protect adenine phosphoribosyltransferase and heated at 60°C for 5 min. This treatment
resulted in fivefold purification and about 95 to 100% of adenine phosphoribosyltransferase activity was recovered. The enzyme preparation was further purified by 80% saturated (NH&SO4 and separated by DEAE-cellulose column. Figure 1 shows that adenine phosphoribosyltransferase was eluted by 0.25 M KC1 in buffer A from DEAE-cellulose column as a single activity peak. To examine if there is an enzyme specific for transferring phosphoribosyl group to cytokinin base, in each step of protein purification Ade was replaced by i’Ade as a substrate for locating the phosphoribosyltransferase activity. The enzyme activity fractions for i’Ade phosphoribosylation were found to be identical to the fractions for Ade. Further purification of the enzyme by Sephadex G-75 column also yeilded a single enzyme activity peak using either [14C]Ade or [14C]i6Adeas a substrate (Fig. 2). Sephadex G-200 chromatography (Fig. 3) resulted in an additional 1.2-fold protein purification. The degree of purification (41.3 nmol/min/mg protein) was approximately 20’7-fold when compared to the crude extract (20,000 g supernatant). The purified enzyme contains no detectable phosphatase activity (10). The molecular weight of 23,000 + 500 for the partially purified enzyme was estimated by gel filtration on Sephadex G-200 column (2.5 X 35 cm). The molecular weight was calculated from standard marker proteins of known molecular weights: horse
CYTOKININ
637
BASE PHOSPHORIBOSYLATION
20 2
4.0
al 5 k
H 3.0
16 E” \ .c E
E
12
2
12.0
E
3 :
r Sk > F
B
s:
P 1.0 4w
I z 0.0
0
50
100
05
150
FRACTION
200
250
300
(1 ml each)
NUMBER
FIG. 1. DEAE-cellulose column chromatography of adenine phosphoribosyltransferase from wheat germ cells. Protein solution (8 ml, 207 mg protein fractionated by 80% ammonium sulfate) was applied to a column (2.5 X 28 cm) preequilibrated with buffer A (50 mM Tris-HCl, pH 7.5; 5 mM MgClz; and 1 mM mercaptoethanol). The column was eluted stepwisely with 96 ml of the same buffer, followed by 0.25 and 0.5 M KC1 in the buffer. One hundred microliters of each fraction was used to measure enzyme activity. Conditions for enzyme activity assays are described in the text. Substrates used were Ade (0) and i’Ade (A).
heart cytochrome c, 13,400; myoglobin, 17,000; ovalbumin, 45,500; bovine plasma albumin, 66,000; and alkaline phosphatase, 77,500.
Optimum
pH
Adenine phosphoribosyltransferase had a pH optimum about 7.5 with either Ade
2 P) 5
1.5 WE % = 5 1.0
40
E 2 .E
302
5”
S z ii !I-
20 z 5 F 10 y
0.5
ii
0.0 C
100
50
FRACTION
NUMBER
150
(3ml each)
OE
z
Y
FIG. 2. Purification of adenine phosphoribosyltransferase by Sephadex G-75 column filtration. The pooled active fraction of adenine phosphoribosyltransferase (28 mg protein in ‘7 ml) obtained from DEAE-cellulose chromatography was applied onto a Sephadex G-75 column (2.5 x 31 cm) preequilibrated with buffer A. The column was eluted with the same buffer. Substrates used were Ade (0) and i6Ade (A). Enzyme activity assays are described in the text.
CHEN, MELITZ, AND CLOUGH
FRACTION
NUMBER (22ml each)
FIG. 3. Sephadex G-ZOOcolumn filtration of adenine phosphoribosyltransferase. The enzyme fraction (5.6 mg protein in 5 ml) obtained from Sephadex G-75 column chromatography was applied onto a column (2.5 X 35 cm) equilibrated with buffer A and the enzyme was eluted with the same buffer. Substrates used were Ade (O), i’Ade (A), and hypoxanthine (m). Enzyme activity assays are described in the text.
or i’Ade as a substrate. These data were obtained with 50 mM Tris-citrate buffer at pH’s from 5 to 7 and 50 ITIM Tris-HCl buffer at pH’s from 7 to 10. The results are illustrated in Fig. 4 (only the data of i’Ade are shown). Phosphwibosylation
Time Course
The time course studies indicated that the rate of i’Ade phosphoribosylation by adenine phosphoribosyltransferase reach a maximum in 15 min at 37°C and level off (Fig. 5). The linearity of i’Ade phosphoribosylation with respect to enzyme concentration is shown in the inset of Fig. 5. Identical results were obtained when Ade was used as a substrate. Effect of Sufiydryl Metal Ions
The enzyme required Mp for full activity. In the presence of 5 mM EDTA without addition of Mg2f, the phosphoribosylation of Ade or i’Ade was abolished. The optimum Mgz+ concentration for the enzyme activity was found to be 5 mM under the standard assay condition using either [8-14C]Ade or [8-14C]i6Ade as a substrate. The effects of chloride form of 5 mM K+,
Reagents and
The enzyme activity was determined in the presence and absence of 0.1 and 1 mM p-chloromercuribenzoate and sodium iodoacetate. There was 63 and 95% inhibition of PAde phosphoribosylation in the presence of 0.1 and 1 mM of p-chloromercuribenzoate, respectively, while 77 and 100% inhibition was observed in the presence of 0.1 and 1 mM of sodium iodoacetate.
PH
FIG. 4. Effect of pH on i’Ade phosphoribosylation of adenine phosphoribosyltransferase. Standard assay conditions were used except that the pH’s of the buffers were varied. Tris-citrate 50 mM (0); 50 mM Tris-HC1 (A).
CYTOKININ
639
BASE PHOSPHORIBOSYLATION TABLE
III
KINETIC CONSTANTS FORADENINE PHOSPHORIBOS~LTRANSFERASE” Kl
V (nmol/min/
Compound
(MM)
mg protein)
Ade i”Ade A?-Fufuryladenine A@-Benzyladenine PRPP
74 130 110 154 15
41 34 26 31 13
V/K,
0.55 0.26 0.23 0.20
aExperimental conditionswere as describedin the text. TlME(min)
FIG. 5. Time course of i’Ade phosphoribosylation by adenine phosphoribosyltransferase. Reactions were carried out under standard assay conditions except incubation times were varied. The effect of protein concentration on i’Ade phosphoribosylation is shown in the inset.
Ca2+ Mn’+, Naf, and Hg2f metal ions on the ‘phosphoribosylation of i’Ade were studied in the presence and absence of 5 mM M2+ in the reaction mixture (Table II). Potassium, manganese, or calcium ions did not have marked effect on the activity of the enzyme in the presence of 5 mM Mg2+. Mn2+ and Ca2+ supported 80 and 54%, respectively, increase in the initial rate of enzyme activity in the absence of Me. The enzyme activity was completely inhibited by H$+. TABLE
II
EFFECT OF METAL IONS ON i’Ade PHOSPHORIBOS~ATION BY ADENINE PHOSPHORIBOWLTRANSFERASE~ Enzyme activity (nmol PAdo-5’-P formed/min/mg protein) Addition (5 mM) None Mn2+ CaZ+ HI2 K+
Without 1.2 17.4 11.8 0 4.5
M$
With 5 mM M2 21.7 22.3 19.4 0 20.6
“The assay conditions were as described in the text. The paper chromatography assay system was employed.
Identi&ation Metabolites
of Phosphoribosylated
Reaction products were separated by paper electrophoresis. The presumed phosphoribosylated radioactive Ade or i’Ade was eluted with water from the electropherogram. The eluate was treated with 2 units (0.1 mg protein) of 5’-nucleotidase of C. adamanteus venom in 0.05 M Tris-HCl buffer (pH 8.5) at 37°C for 30 min. After this treatment the resulting product migrated with the corresponding unlabeled nucleoside marker in paper electrophoretic analysis. These results indicated that the nucleotide was the 5’-monophosphate. Alternatively, larger quantities of dephosphorylated nucleotide products were obtained by scaling up of experiments using 14C-labeled compounds and replacing them with unlabeled ones. The uv absorption spectra of the purified unlabeled dephosphorylated products were: i’Ado: X,,, at pH 2, 265 nm; at pH 7, 269 nm; at pH 12, 269 nm; and Ado: at pH 2, 257 nm; at pH 7,260 nm; at pH 12,259 nm. These values agree with the values of corresponding authentic compounds. Kinetic
Studies
A kinetic study of the phosphoribosylation of various cytokinins was carried out as described under Materials and Methods using the enzyme from Sephadex G-200 chromatography, and comparisons with Ade were made (Table III). The K, and V were calculated from LineweaverBurke plots with data from at least seven
640
CHEN. MELITZ,
different cytokinin base concentrations (1 X lo-’ to 1 X 1O-4M) in the presence of 600 FM PRPP. It is apparent from Table III that the affinity of fl-furfuryladenine and A@-benzyladenine for the adenine phosphoribosyltransferase approximates that of i’Ade. However, Ade had about 50 to 100% higher affinity for the enzyme than these cytokinins. The V/K, ratio indicates that Ade is about 2.1- to 2.7-fold more efficient as a substrate than the cytokinins for the enzyme. Substrate specificity studies indicated that neither guanine nor hypoxanthine served as a substrate for this enzyme. DISCUSSION
The results reported here show that partially purified adenine phosphoribosyltransferase from wheat germ catalyzes the conversion of cytokinin bases as well as Ade to form corresponding nucleotides. Phosphoribosylation of i’Ade and Ade may be catalyzed by this same enzyme because adenine phosphoribosyltransferase was the only enzyme detected capable of phosphoribosylating the cytokinin bases and Ade (Figs. 1,2, and 3), and these substrates also had the same pH optimum (7.5). This enzyme is not a hypoxanthineguanine phosphoribosyltransferase (EC 2.4.2.8) because hypoxanthine or guanine did not serve as a substrate. The reported Michaelis constants of adenine phosphoribosyltransferase from various animal tissues ranged from 0.6 to 140 PM for Ade (13-15). The enzyme from soybean callus was reported to have Michaelis constant for Ade of 1.5 pM (25), and the presence of a cytokinin increased the activity of this enzyme in extracts of soybean callus and senescing barley leaves. With the wheat germ enzyme, Km value for Ade was ‘74 pM, and for the tested cytokinin bases ranging from 110 to 154 pM (Table III). It is obvious that replacement of the N6-amino group of Ade by an isopentenyl, furfuryl, or benzyl amino side chain increases the Km values of the reaction by factors of 1.5 to 2.1 (Table III). Furthermore, the ratio of V:K, indicates that Ade is about 2.1- to 2.8-fold more ef-
AND CLOUGH
ficient than the cytokinin bases as a substrate. This may help to explain why free Ade virtually does not exist in tissues (26), but free cytokinin bases can be found in various plant tissues (l-7). Cytokinin regulation of physiological processes in a plant system is accomplished by a variety of mechanisms. For example, the plant tissues’ response to cytokinin is proportional to the concentration of the hormone present; higher concentrations are inhibitory, whereas removal of this hormone diminishes or eliminates the response. Thus, by regulating the rate of cytokinin metabolism the tissue responses to cytokinin may be controlled. Consequently, the cytokinin metabolic enzymes, such as adenine phosphoribosyltransferase, may play an important role in balancing the supply of “active cytokinin.” REFERENCES 1. HALL, R. H. (1970) in Progress in Nucleic Acid Research and Molecular Biology (Davidson, W. E., and Cohn, W. E., eds.), Vol. 10, pp. 5786, Academic Press, New York. 2. FOX, J. E. (1969) in Physiology of Plant Growth and Development (Wilkins, M. B., ed.), pp. 8% 123, McGraw-Hill, New York. 3. SKOOC, G., AND ARMSTRONG, D. J. (1970) Annu. Rev. Plant Physiol 21, 359-384. 4. CHEN, C. M., AND PETSCHOW, B. (1978) Plant PhysioL 62, 861-865. 5. EINSET, J. W., AND SKOOG, F. (1973) Proc. Nat. Acad Sci. USA 70, 658-660. 6. LALOUE, M., TERRINE, C., AND GAWER, M. (1974) FEBS Let.?. 46, 45-49. 7. MILLER, C. 0. (1975) Plant PhysioL 55, 448-449. 8. CHEN, C. M., AND PETSCHOW, B. (1978) Plant PhysioL 62, 871-874. 9. CHEN, C. M., AND MELITZ, D. K. (1970) FEBS Lett. 107, 15-20. 10. CHEN, C. M., AND KRISTOPEIT, S. M. (1981) Plant PhysioL 67, 494-498. 11. LALOUE, M., TERRINE, C., AND GUERN, J. (1977) Plant PhysioL 59, 478-483. 12. DOREE, M., AND GUERN, J. (1973) B&him Bitphys. Acta 304, 611-622. 13. THOMAS, C. B., ARNOLD, W. J., AND KELLEY, W. N. (1973) J. BioL Chem 248, 2529-2535. 14. KENIMER, J. G., YOUNG, L. G., AND GROTH, D. P. (1975) Biochim Biophys. Acta 384, 87-101. 15. HOLDEN, J. A., MEREDITH, G. S., AND KELLEY, W. N. (1979) .I BioL Chem. 254, 6951-6955.
CYTOKININ
BASE PHOSPHORIBOSYLATION
16. BERLIN, R. D. (1969) Arch. Biochem. Biophys. 134, 120-129. 17. ROY-BURMAN, S., AND VISSER, D. W. (1975) J. Biol Chem 250, 9270-9275. 18. NAGY, M., AND RIBET, A. (1977) Eur. J Biochem. 77, 77-85. 19. BRADFORD, M. M. (1976) Anal. Biochem 72,248254. 20. BRAY, G. (1960) Anal. Biochem. 1, 279-285. 21. CHEN, C. M., MELITZ, D. K., PETSCHOW, B., AND ECKERT, R. L. (1980) Eur. J. B&hem. 108,379387. 22. HALL, R. H., AND ROBINS, M. J. (1968) in Syn-
23.
24. 25. 26.
641
thetic Procedures in Nucleic Acid Chemistry (Zorbach, W. W., and Tipson, R. S., eds.), pp. H-13, Interscience-Wiley, New York. HECHT, S. M., HELGESON, J. P., AND FUJII, T. (1968) in Synthetic Procedures in Nucleic Acid Chemistry (Zorbach, W. W., and Tipson, R. S., eds.), pp. 8-10, Interscience-Wiley, New York. BENDICH, A., RUSSEL, P. J., AND Fox, J. J. (1954) J. Amer. Chem Sot. 76, 6073-6077. NICHOLLS, P. B., AND MURRAY, A. W. (1968) Plant PhysioL 43, 645-648. HENDERSON, J. F., AND PATERSON, A. R. P. (1973) Nucleotide Metabolism, pp. 126-129, Academic Press, New York.
Metabolism of Cytokinin: Phosphoribosylation of Cytokinin Bases by Adenine Phosphoribosyltransferase from Wheat Germ’ CHONG-MAW CHEN,’ DEBRA K. MELITZ, AND FRED W. CLOUGH Department
of Life Science and Biomedical Research Institute, University Kenosha, Wisconsin 53161 Received July 15, 1981, and in revised form November
of Wisconsin-Pa&side, 13, 1981
Adenine phosphoribosyltransferase (AMP:pyrophosphate phosphoribosyltransferase EC 2.4.2.8) which catalyzes the phosphoribosylation of cytokinin bases and adenine to form the corresponding nucleotides were partially purified from the cytosol of wheat (Triticum aestivum) germ. This enzyme (molecular weight, 23,000 + 500) phosphoribosylates the bases at an optimum Mg2+ concentration of 5 MM and optimum pH of 7.5 (50 mM Tris-HCl buffer). K, values for NG-(A’-isopentenyl)adenine, NG-furfuryladenine, NG-benzyladenine, and adenine are 130, 110, 154, and 74 PM, respectively, in 50 mM Tris-HCl buffer (pH 7.5) at 37°C. Hypoxanthine and guanine are not substrates for the enzyme. In concerting with other cytokinin metabolic enzymes, this enzyme may play a significant role in maintaining the supply of adequate levels of “active cytokinin.”
Cytokinins are a group of plant hormones which regulate cell division and differentiation (l-3). Cytokinin bases and cytokinin ribonucleotides have been found in various plant cells, and these cytokinins are metabolized in the plant cell to form different metabolites (4-11). The relative amount of a specific metabolite varies in different tissues under different physiological conditions. Doree and Guern (12) studied short-term metabolism of some exogenous cytokinins in Acer peudoplatanus cells and observed that one of the major metabolites formed from cytokinin base was the corresponding nucleotide. Their observation suggests that in plant cells there is an enzyme system catalyzing the formation of cytokinin ribonucleotides from their corresponding bases. Although
phosphoribosylation of Ade to form AMP by adenine phosphoribosyltransferase has been widely studied in animal (13-15) and microbial (16-18) cells, there have been very few studies of this enzyme from plant cells; furthermore, the role of this enzyme in cytokinin metabolism in plant cells has not been defined. We report here the partial purification of adenine phosphoribosyltransferase from wheat germ, the properties of the enzyme, and the kinetics of the phosphoribosylation of cytokinin bases by this enzyme. MATERIALS
Chemicals and Enzymes Ade, AMP, i6Ade,3 NG-benzyladenine, NG-furfuryladenine, or kinetin, other ribonucleotides, Carbowax (Mr wt., 6000), 5’-nucleotidase (crotalus adaman-
1 This work was supported by the National Science Foundation Research Grant PCM 79 03832 to C.-M.C. ZAuthor to whom all correspondence should be addressed. 0003-9861/82/040634-08$02.00/O Copyright All rights
0 1982 by Academic Press, Inc. of reproduction in any form reserved.
AND METHODS
3 Abbreviations used: PRPP: 5-phosphoribosyl-lpyrophosphate; i’Ade: fl-(A*-isopentenyl)adenine; i’Ado: fl-(A’-isopentenyl)adenosine; P, phosphate. 634
CYTOKININ BASE PHOSPHORIBOSYLATION teus venom), 5-phosphoribosyl-1-pyrophosphate, and wheat (Triticum aestivum) germ were from Sigma Chemical Company; i’Ado-5’-P was from P-L. Biochemical Company; [8-‘4C]hypoxanthine (62 &i/p mol), N6[8-i4C]furfuryladenine (15 &i/pmol), and N6[8-i4C]benzyladenine (13.4 &i/pmol) were from Amersham Corporation.
Analytical
Methods
Protein concentration was determined according to the method of Bradford (19) using bovine serum albumin as a standard. A Cary Model 14 spectrophotometer was used to quantify purine bases and nucleotides. Nucleotides, nucleosides, and bases were separated by paper electrophoresis (Camag TLE Cell, Whatman No. 3MM paper) with 0.1 M Tris-citrate buffer (pH 3.5) at 20°C and/or paper chromatography (Whatman No. 3MM) in a descending fashion using the following solvent system (v/v): A, ethylacetate:l-propanol:HzO (4:1:2), B, 1-butanol: HzO:concentrated NH,OH (86:14:5); C, 2-propanol: HzO:concentrated NH,OH (721); D, 95% ethano1:O.l M (NH&B03 (pH 9.0) (1:9), E, 1-propanol:concentrated NHIOH:HzO (60:20:20).Sephadex LH-20 resin (Pharmacia) was swollen in 35% aqueous ethanol and the column was eluted with this solvent. Radioactivity was measured in a Tracer Analytic Mark III liquid scintillation system. For liquid samples, an aliquot of no more than 0.5 ml was added to 10 ml of Bray’s solution (20). For paper chromatograms, l- or 2-cm sections were placed in vials containing scintillation fluid (21).
Preparation
of [8’4C]i6Ade
[8-‘4C]i6Ade was synthesized from 3-methy -2-butenylamine (hydrochloride) and 6-[8-‘4C]chloropurine (22). The former compound was prepared according to the procedure of Hecht et al. (23). 6-[B‘*C]Chloropurine, commercially not available, was synthesized from [S-“Clhypoxanthine by the method of Bendich et al. (24). The synthesized [8-‘*C]i6Ade was purified by Sephadex LH-20 column and then by paper chromatography in solvent system C. The synthesized radioactive i’Ade had uv absorption spectra Amax272 nm at pH 2; 269 nm at pH 7, and 275 nm at pH 12. Paper chromatographic mobility of [8-14C]i6Ade was corresponded to that of authentic i’Ade in five solvent systems (A to E).
Extraction
and Fractionation
of Enzyme
Wheat germ (200 g) frozen with liquid N2 was homogenized in a Waring Blendor in 4 vol/weight of buffer A (50 mM Tris-HCl, pH 7.5, 5 mM MgCla, and 1 mM mercaptoethanol). The homogenate was centrifuged for 10 min at 15,000~ and resulting super-
635
natant was centrifuged again for 30 min at 20,OOOg. The supernatant is referred to as crude extract. The following steps were employed to purify the extract further. All procedures were conducted at 0 to 4°C. AMP and (NH&SO4 were Step 1: Heat treatment. added to the crude extract to a final concentration of 1 and 30 mM, respectively, and the mixture was heated for 5 min in water bath at 60°C. After cooling to 4°C in an ice bath, it was centrifuged for 30 min at 20,OOOgand the precipitate was discarded. Step 2: Ammonium sulfate fractionation. Solid ammonium sulfate was added to the collected supernatant to make 30% saturation. After 1 h the sample was centrifuged at 20,OOOgfor 20 min. The 30% supernatant was brought to 80% saturation by addition of solid ammonium sulfate, and the precipitate was again collected by centrifugation at 20,OOOgfor 20 min. The pellet was resuspended in buffer A and dialyzed for 18 h against buffer A. The dialysate was reduced to 8 ml by Carbowax. Step 3: DEAE-cellulose chromatography. The concentrated protein solution (207 mg protein) was applied onto a DEAE-cellulose (Whatman DE-23) column (2.5 X 28 cm) equilibrated with buffer A. The column was eluted with 0.72 bed vol of this buffer, followed by the same bed volumes of 0.25 M KC1 in buffer A, and finally eluted with 0.5 M KC1 in this buffer. The adenine phosphoribosyltransferase fractions were pooled and dialyzed against buffer A for 17 h. The protein solution was reduced to 7 ml by Carbowax. Step 4: Sephadex G-75$ltration. The enzyme solution (28 mg protein) was filtered through a Sephadex G-75 column (2.5 X 31 cm) and eluted with buffer A. The fractions containing adenine phosphoribosyltransferase activity were pooled and reduced to 5 ml. Step 5: Sephadex G-.%Nchnnnatography. The Sephadex G-75 fraction (5.6 mg protein) was further chromatographed on Sephadex G-200 column (2.5 X 35 cm) equilibrated with buffer A. The enzyme was eluted with the same buffer and the active enzyme fractions were pooled, reduced to 10 ml by Carbowax, and stored at -20°C.
Enzyme Assays The adenine phosphoribosyltransferase activity was measured either by paper chromatographic method or by butanol extraction method. The first method depended on direct measurement of radioactivity of reaction product after chromatographic separation. The second method is based on the differential solubility of substrate and reaction product. In the first assay method, the reaction mixture (170 ~1) contained 36 PM [8-14C]Ade,or 32 pM [8-i4C]i6Ade, 5 mM MgClz, 1 mM fl-mercaptoethanol, 600 pM PRPP, 50 mM Tris-HCl buffer (pH 7.5), and the enzyme (1560 pg protein). The reaction was started by adding
636
CHEN, MELITZ, AND CLOUGH TABLE I PURIFICATION OF WHEAT GERM ADENINE PHOSPHORIBOSYL TRANSFERASE’
Fraction
Total protein (mg)
1. 20,000g supernatant 2. Heat treatment 3. 80% (NH&SO1 4. DEAE-cellulose pool 5. Sephadex G-75 pool 6. Sephadex G-200 pool
4955 902 207 28 5.6 2.4
Specific activity (nmol/min/mg) 0.2 1.0 3.3 11.9 33.5 41.3
Total activity (nmol/min) 991 902 683 333 188 99
Recovery (%I 100 91 69 34 19 10
Purification (n-fold) (1.0) 5 17 60 168 207
a The starting material was 200 g wheat germ cells. Enzyme activity was determined as described in the text with Ade as a substrate. The paper chromatographic assay system was used. the enzyme to the reaction mixture which had been warmed at 37°C. After incubation for 15 min at 37”C, the reaction was stopped by the addition of 100 11 of 95% ethanol. The mixture was spotted on Whatman No. 3MM paper with AMP or i’Ado-5’-P as carrier, and the chromatogram was developed with solvent D. In the second assay method, the reaction mixture containing components identical to the first method was incubated for 15 min at 3’7°C. At the end of the reaction, 2 ml of water-saturated butanol was added to the reaction mixture and the content was mixed thoroughly. The upper butanol phase was carefully siphoned from the lower water phase. This butanol extraction procedure was repeated eight times. About 90% of Ade was found in the butanol phase. The water phase containing [8-14C]AMP and less than 10% of Ade was transferred to scintillation vial and the radioactivity was determined. The background counts (10%) were substrated from the total counts. This butanol extraction method was used only in locating the enzyme fractions eluted from column chromatographies. Furthermore, since cytokinin nucleotide is soluble both in butanol and water, this method was not employed for cytokinin nucleotide synthesis assay. RESULTS
Pur$icatim of Adenine Phosphtibosyltransferase
Purification of this enzyme from 200 g of wheat germ cells is summarized in Table I. In the heat treatment step, 30 mM (NH&SO4 and 1 mM AMP were added to the crude enzyme extract to protect adenine phosphoribosyltransferase and heated at 60°C for 5 min. This treatment
resulted in fivefold purification and about 95 to 100% of adenine phosphoribosyltransferase activity was recovered. The enzyme preparation was further purified by 80% saturated (NH&SO4 and separated by DEAE-cellulose column. Figure 1 shows that adenine phosphoribosyltransferase was eluted by 0.25 M KC1 in buffer A from DEAE-cellulose column as a single activity peak. To examine if there is an enzyme specific for transferring phosphoribosyl group to cytokinin base, in each step of protein purification Ade was replaced by i’Ade as a substrate for locating the phosphoribosyltransferase activity. The enzyme activity fractions for i’Ade phosphoribosylation were found to be identical to the fractions for Ade. Further purification of the enzyme by Sephadex G-75 column also yeilded a single enzyme activity peak using either [14C]Ade or [14C]i6Adeas a substrate (Fig. 2). Sephadex G-200 chromatography (Fig. 3) resulted in an additional 1.2-fold protein purification. The degree of purification (41.3 nmol/min/mg protein) was approximately 20’7-fold when compared to the crude extract (20,000 g supernatant). The purified enzyme contains no detectable phosphatase activity (10). The molecular weight of 23,000 + 500 for the partially purified enzyme was estimated by gel filtration on Sephadex G-200 column (2.5 X 35 cm). The molecular weight was calculated from standard marker proteins of known molecular weights: horse
CYTOKININ
637
BASE PHOSPHORIBOSYLATION
20 2
4.0
al 5 k
H 3.0
16 E” \ .c E
E
12
2
12.0
E
3 :
r Sk > F
B
s:
P 1.0 4w
I z 0.0
0
50
100
05
150
FRACTION
200
250
300
(1 ml each)
NUMBER
FIG. 1. DEAE-cellulose column chromatography of adenine phosphoribosyltransferase from wheat germ cells. Protein solution (8 ml, 207 mg protein fractionated by 80% ammonium sulfate) was applied to a column (2.5 X 28 cm) preequilibrated with buffer A (50 mM Tris-HCl, pH 7.5; 5 mM MgClz; and 1 mM mercaptoethanol). The column was eluted stepwisely with 96 ml of the same buffer, followed by 0.25 and 0.5 M KC1 in the buffer. One hundred microliters of each fraction was used to measure enzyme activity. Conditions for enzyme activity assays are described in the text. Substrates used were Ade (0) and i’Ade (A).
heart cytochrome c, 13,400; myoglobin, 17,000; ovalbumin, 45,500; bovine plasma albumin, 66,000; and alkaline phosphatase, 77,500.
Optimum
pH
Adenine phosphoribosyltransferase had a pH optimum about 7.5 with either Ade
2 P) 5
1.5 WE % = 5 1.0
40
E 2 .E
302
5”
S z ii !I-
20 z 5 F 10 y
0.5
ii
0.0 C
100
50
FRACTION
NUMBER
150
(3ml each)
OE
z
Y
FIG. 2. Purification of adenine phosphoribosyltransferase by Sephadex G-75 column filtration. The pooled active fraction of adenine phosphoribosyltransferase (28 mg protein in ‘7 ml) obtained from DEAE-cellulose chromatography was applied onto a Sephadex G-75 column (2.5 x 31 cm) preequilibrated with buffer A. The column was eluted with the same buffer. Substrates used were Ade (0) and i6Ade (A). Enzyme activity assays are described in the text.
CHEN, MELITZ, AND CLOUGH
FRACTION
NUMBER (22ml each)
FIG. 3. Sephadex G-ZOOcolumn filtration of adenine phosphoribosyltransferase. The enzyme fraction (5.6 mg protein in 5 ml) obtained from Sephadex G-75 column chromatography was applied onto a column (2.5 X 35 cm) equilibrated with buffer A and the enzyme was eluted with the same buffer. Substrates used were Ade (O), i’Ade (A), and hypoxanthine (m). Enzyme activity assays are described in the text.
or i’Ade as a substrate. These data were obtained with 50 mM Tris-citrate buffer at pH’s from 5 to 7 and 50 ITIM Tris-HCl buffer at pH’s from 7 to 10. The results are illustrated in Fig. 4 (only the data of i’Ade are shown). Phosphwibosylation
Time Course
The time course studies indicated that the rate of i’Ade phosphoribosylation by adenine phosphoribosyltransferase reach a maximum in 15 min at 37°C and level off (Fig. 5). The linearity of i’Ade phosphoribosylation with respect to enzyme concentration is shown in the inset of Fig. 5. Identical results were obtained when Ade was used as a substrate. Effect of Sufiydryl Metal Ions
The enzyme required Mp for full activity. In the presence of 5 mM EDTA without addition of Mg2f, the phosphoribosylation of Ade or i’Ade was abolished. The optimum Mgz+ concentration for the enzyme activity was found to be 5 mM under the standard assay condition using either [8-14C]Ade or [8-14C]i6Ade as a substrate. The effects of chloride form of 5 mM K+,
Reagents and
The enzyme activity was determined in the presence and absence of 0.1 and 1 mM p-chloromercuribenzoate and sodium iodoacetate. There was 63 and 95% inhibition of PAde phosphoribosylation in the presence of 0.1 and 1 mM of p-chloromercuribenzoate, respectively, while 77 and 100% inhibition was observed in the presence of 0.1 and 1 mM of sodium iodoacetate.
PH
FIG. 4. Effect of pH on i’Ade phosphoribosylation of adenine phosphoribosyltransferase. Standard assay conditions were used except that the pH’s of the buffers were varied. Tris-citrate 50 mM (0); 50 mM Tris-HC1 (A).
CYTOKININ
639
BASE PHOSPHORIBOSYLATION TABLE
III
KINETIC CONSTANTS FORADENINE PHOSPHORIBOS~LTRANSFERASE” Kl
V (nmol/min/
Compound
(MM)
mg protein)
Ade i”Ade A?-Fufuryladenine A@-Benzyladenine PRPP
74 130 110 154 15
41 34 26 31 13
V/K,
0.55 0.26 0.23 0.20
aExperimental conditionswere as describedin the text. TlME(min)
FIG. 5. Time course of i’Ade phosphoribosylation by adenine phosphoribosyltransferase. Reactions were carried out under standard assay conditions except incubation times were varied. The effect of protein concentration on i’Ade phosphoribosylation is shown in the inset.
Ca2+ Mn’+, Naf, and Hg2f metal ions on the ‘phosphoribosylation of i’Ade were studied in the presence and absence of 5 mM M2+ in the reaction mixture (Table II). Potassium, manganese, or calcium ions did not have marked effect on the activity of the enzyme in the presence of 5 mM Mg2+. Mn2+ and Ca2+ supported 80 and 54%, respectively, increase in the initial rate of enzyme activity in the absence of Me. The enzyme activity was completely inhibited by H$+. TABLE
II
EFFECT OF METAL IONS ON i’Ade PHOSPHORIBOS~ATION BY ADENINE PHOSPHORIBOWLTRANSFERASE~ Enzyme activity (nmol PAdo-5’-P formed/min/mg protein) Addition (5 mM) None Mn2+ CaZ+ HI2 K+
Without 1.2 17.4 11.8 0 4.5
M$
With 5 mM M2 21.7 22.3 19.4 0 20.6
“The assay conditions were as described in the text. The paper chromatography assay system was employed.
Identi&ation Metabolites
of Phosphoribosylated
Reaction products were separated by paper electrophoresis. The presumed phosphoribosylated radioactive Ade or i’Ade was eluted with water from the electropherogram. The eluate was treated with 2 units (0.1 mg protein) of 5’-nucleotidase of C. adamanteus venom in 0.05 M Tris-HCl buffer (pH 8.5) at 37°C for 30 min. After this treatment the resulting product migrated with the corresponding unlabeled nucleoside marker in paper electrophoretic analysis. These results indicated that the nucleotide was the 5’-monophosphate. Alternatively, larger quantities of dephosphorylated nucleotide products were obtained by scaling up of experiments using 14C-labeled compounds and replacing them with unlabeled ones. The uv absorption spectra of the purified unlabeled dephosphorylated products were: i’Ado: X,,, at pH 2, 265 nm; at pH 7, 269 nm; at pH 12, 269 nm; and Ado: at pH 2, 257 nm; at pH 7,260 nm; at pH 12,259 nm. These values agree with the values of corresponding authentic compounds. Kinetic
Studies
A kinetic study of the phosphoribosylation of various cytokinins was carried out as described under Materials and Methods using the enzyme from Sephadex G-200 chromatography, and comparisons with Ade were made (Table III). The K, and V were calculated from LineweaverBurke plots with data from at least seven
640
CHEN. MELITZ,
different cytokinin base concentrations (1 X lo-’ to 1 X 1O-4M) in the presence of 600 FM PRPP. It is apparent from Table III that the affinity of fl-furfuryladenine and A@-benzyladenine for the adenine phosphoribosyltransferase approximates that of i’Ade. However, Ade had about 50 to 100% higher affinity for the enzyme than these cytokinins. The V/K, ratio indicates that Ade is about 2.1- to 2.7-fold more efficient as a substrate than the cytokinins for the enzyme. Substrate specificity studies indicated that neither guanine nor hypoxanthine served as a substrate for this enzyme. DISCUSSION
The results reported here show that partially purified adenine phosphoribosyltransferase from wheat germ catalyzes the conversion of cytokinin bases as well as Ade to form corresponding nucleotides. Phosphoribosylation of i’Ade and Ade may be catalyzed by this same enzyme because adenine phosphoribosyltransferase was the only enzyme detected capable of phosphoribosylating the cytokinin bases and Ade (Figs. 1,2, and 3), and these substrates also had the same pH optimum (7.5). This enzyme is not a hypoxanthineguanine phosphoribosyltransferase (EC 2.4.2.8) because hypoxanthine or guanine did not serve as a substrate. The reported Michaelis constants of adenine phosphoribosyltransferase from various animal tissues ranged from 0.6 to 140 PM for Ade (13-15). The enzyme from soybean callus was reported to have Michaelis constant for Ade of 1.5 pM (25), and the presence of a cytokinin increased the activity of this enzyme in extracts of soybean callus and senescing barley leaves. With the wheat germ enzyme, Km value for Ade was ‘74 pM, and for the tested cytokinin bases ranging from 110 to 154 pM (Table III). It is obvious that replacement of the N6-amino group of Ade by an isopentenyl, furfuryl, or benzyl amino side chain increases the Km values of the reaction by factors of 1.5 to 2.1 (Table III). Furthermore, the ratio of V:K, indicates that Ade is about 2.1- to 2.8-fold more ef-
AND CLOUGH
ficient than the cytokinin bases as a substrate. This may help to explain why free Ade virtually does not exist in tissues (26), but free cytokinin bases can be found in various plant tissues (l-7). Cytokinin regulation of physiological processes in a plant system is accomplished by a variety of mechanisms. For example, the plant tissues’ response to cytokinin is proportional to the concentration of the hormone present; higher concentrations are inhibitory, whereas removal of this hormone diminishes or eliminates the response. Thus, by regulating the rate of cytokinin metabolism the tissue responses to cytokinin may be controlled. Consequently, the cytokinin metabolic enzymes, such as adenine phosphoribosyltransferase, may play an important role in balancing the supply of “active cytokinin.” REFERENCES 1. HALL, R. H. (1970) in Progress in Nucleic Acid Research and Molecular Biology (Davidson, W. E., and Cohn, W. E., eds.), Vol. 10, pp. 5786, Academic Press, New York. 2. FOX, J. E. (1969) in Physiology of Plant Growth and Development (Wilkins, M. B., ed.), pp. 8% 123, McGraw-Hill, New York. 3. SKOOC, G., AND ARMSTRONG, D. J. (1970) Annu. Rev. Plant Physiol 21, 359-384. 4. CHEN, C. M., AND PETSCHOW, B. (1978) Plant PhysioL 62, 861-865. 5. EINSET, J. W., AND SKOOG, F. (1973) Proc. Nat. Acad Sci. USA 70, 658-660. 6. LALOUE, M., TERRINE, C., AND GAWER, M. (1974) FEBS Let.?. 46, 45-49. 7. MILLER, C. 0. (1975) Plant PhysioL 55, 448-449. 8. CHEN, C. M., AND PETSCHOW, B. (1978) Plant PhysioL 62, 871-874. 9. CHEN, C. M., AND MELITZ, D. K. (1970) FEBS Lett. 107, 15-20. 10. CHEN, C. M., AND KRISTOPEIT, S. M. (1981) Plant PhysioL 67, 494-498. 11. LALOUE, M., TERRINE, C., AND GUERN, J. (1977) Plant PhysioL 59, 478-483. 12. DOREE, M., AND GUERN, J. (1973) B&him Bitphys. Acta 304, 611-622. 13. THOMAS, C. B., ARNOLD, W. J., AND KELLEY, W. N. (1973) J. BioL Chem 248, 2529-2535. 14. KENIMER, J. G., YOUNG, L. G., AND GROTH, D. P. (1975) Biochim Biophys. Acta 384, 87-101. 15. HOLDEN, J. A., MEREDITH, G. S., AND KELLEY, W. N. (1979) .I BioL Chem. 254, 6951-6955.
CYTOKININ
BASE PHOSPHORIBOSYLATION
16. BERLIN, R. D. (1969) Arch. Biochem. Biophys. 134, 120-129. 17. ROY-BURMAN, S., AND VISSER, D. W. (1975) J. Biol Chem 250, 9270-9275. 18. NAGY, M., AND RIBET, A. (1977) Eur. J Biochem. 77, 77-85. 19. BRADFORD, M. M. (1976) Anal. Biochem 72,248254. 20. BRAY, G. (1960) Anal. Biochem. 1, 279-285. 21. CHEN, C. M., MELITZ, D. K., PETSCHOW, B., AND ECKERT, R. L. (1980) Eur. J. B&hem. 108,379387. 22. HALL, R. H., AND ROBINS, M. J. (1968) in Syn-
23.
24. 25. 26.
641
thetic Procedures in Nucleic Acid Chemistry (Zorbach, W. W., and Tipson, R. S., eds.), pp. H-13, Interscience-Wiley, New York. HECHT, S. M., HELGESON, J. P., AND FUJII, T. (1968) in Synthetic Procedures in Nucleic Acid Chemistry (Zorbach, W. W., and Tipson, R. S., eds.), pp. 8-10, Interscience-Wiley, New York. BENDICH, A., RUSSEL, P. J., AND Fox, J. J. (1954) J. Amer. Chem Sot. 76, 6073-6077. NICHOLLS, P. B., AND MURRAY, A. W. (1968) Plant PhysioL 43, 645-648. HENDERSON, J. F., AND PATERSON, A. R. P. (1973) Nucleotide Metabolism, pp. 126-129, Academic Press, New York.