Etiolated Mung Bean Seedlings Research Articles

The effect of mung bean seedlings juice (<i>Vigna radiata (L.</i>) Wilczek.) on growth processes and protection against phytopathogens of spring wheat (<I>Triticum aestivum L.</I>) at early stages of organogenesis

Relevance. Mung bean is a new culture of versatile use for the Middle Volga region; it has a complex of valuable economic and biological properties. When studying seeds, seedlings and mung bean plants as raw materials for the pharmaceutical industry, it was revealed that this culture also has a biocidal effect on phytopathogens. The purpose of our research is to study the biological activity of the juice from the sprouts of mung bean on the seedlings of spring soft wheat for the subsequent creation of theoretical foundations for the development and use of mung bean as a growth stimulant and protection of wheat seedlings from phytopathogenic organisms.Methods. To obtain juice, three-day seedlings of mung bean of the Saltan variety were taken. Three varieties of spring soft wheat created at the Volga Research Institute of Breeding and Seed Production named after P.N. Konstantinov were used in the experiments — Kinelskaya 2020, Kinelskaya jubilee, Kinelskaya Zvezda.Results. The effect of juice from green and etiolated mung bean seedlings on phytopathogens and the growth of spring wheat seeds in the early stages of organogenesis was studied. It was found that the juice of green seedlings has a pronounced biocidal and stimulating activity. Thus, in the tested variants, the formation of primary roots exceeded the control by 15–26%, the length of primary roots exceeded the control by 248– 297% and the length of sprouts — by 337–403%. There were no signs of damage in all tested variants. In the juice of etiolated seedlings, such activity is less pronounced. Only at a concentration of cell juice of 1% in the studied wheat varieties, an excess over the control was observed: the formation of primary roots — by106–112%, the length of primary roots — by109–127% and the length of sprouts 108–125%. In other versions of this experiment, the results were ambiguous. The defeat of phytopathogens manifested itself, but the number of affected wheat seeds decreased as the concentration of cell juice solutions increased.

Effects of superoxide radicals on ACC synthase activity in chilling-stressed etiolated mungbean seedlings

The activity of 1-aminocyclopropane-1-carboxylic acid synthase (ACC synthase, ACS) and the concentrations of superoxide radical (O2−.) and hydrogen peroxide (H2O2) were measured in etiolated mungbean seedlings following their transfer to a growth chamber at 25°C after a 5-h-chilling treatment at 5°C. All of these variables increased dramatically after the transfer, and strong correlations were found between ACS activity and the concentrations of superoxide and H2O2. Exogenous applications of two generators of superoxide radicals, methylviologen (MV) and xanthine–xanthine oxidase (X–XOD), enhanced ACS activity in seedlings, but their effects were inhibited by exogenous applications of specific scavengers of O2−.. However, applications of H2O2 or specific H2O2-scavengers had no significant effects on seedlings ACS activity. The results indicate that O2−. was involved in the chilling-induced increases in ACS activity, but not H2O2. ACS activity peaked ca. 8 h after the transfer, and then declined, but the decline could be counteracted by exogenous applications of specific O2−. scavengers, this suggests that damage was caused by superoxide radicals influencing ACS activity in etiolated mungbean seedlings. Further analysis of changes in two key kinetic parameters of ACS activity—Vmax (maximum velocity) and Km (the Michaelis constant)—in the seedlings indicated that the presence of O2−. may reduce Km, i.e. increase substrate (S-adenosyl methionine, SAM) affinity. That would be the main mechanism responsible for the observed chilling-induced increases in ACS activity in etiolated mungbean seedlings.

Differential response of vacuolar proton pumps to osmotica.

The vacuole is a fundamental and dominant organelle and occupies a large part of the total cell volume in most mature plant cells. The higher-plant vacuole contains two types of proton-translocating pumps, H+-ATPase (EC 3.6.1.3) and H+-pyrophosphatase (EC 3.6.1.1), residing on the same membrane. These two enzymes generate roughly equal proton gradients across the vacuolar membrane for the secondary transport of ions and metabolites. However, the pumps respond differentially to stress in order to maintain critical functions of the vacuole. In this work, tonoplasts from etiolated mung bean seedlings (Vigna radiata L.) were used to investigate the function of these two enzymes under high osmotic pressure. At high concentrations of sucrose or sorbitol, the light scattering and volume of isolated vesicles were progressively changed. Concomitantly, enzymatic activities, proton translocation, and coupling efficiencies of these two proton-pumping enzymes were inhibited to various extents under high osmotic pressure. No significant change in enzymatic activities of purified vacuolar H+-PPase and H+-ATPase under similar conditions was observed. We thus believe that the membrane structure is an important determinant for proper function of proton pumping systems of plant vacuoles. Furthermore, kinetic analysis shows different variation in apparent Vmax but not in KM values of vacuolar H+-PPase and H+-ATPase at high osmolarity of sucrose and sorbitol, respectively, suggesting probable alterations in substrate hydrolysis reactions but not substrate-binding affinity of the enzymes. A working model is proposed to interpret supplemental roles of vacuolar H+-PPase and H+-ATPase to maintain appropriate functions of plant tonoplasts.

Developmental regulation of the expression of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase and ACC oxidase genes in hypocotyls of etiolated mung bean seedlings

Differential expression of seven 1-aminocyclopropane-1-carboxylate (ACC) synthase and two ACC oxidase genes during the developmental process was investigated in etiolated mung bean seedlings. Among these genes, VR-ACS1, VR-ACS6, VR-ACS7, VR-ACO1, and VR-ACO2 were expressed in the subhook regions of hypocotyls. Except for VR-ACO1, the expression levels of these genes were correlated with the amount of ethylene produced. VR-ACO1 expression was largely constitutive. During hypocotyl growth, the expression of VR-ACS1, VR-ACS6, and VR-ACS7 was maintained at a high level. After the growth stopped, their expression levels became negligible, but the decrease in the VR-ACO2 transcript was much slower. These results suggest that the expression of each of the five genes in the subhook regions of hypocotyls is kinetically distinct, temporally and spatially regulated, and especially high in the tissues of rapidly elongating cells. However, endogenously produced ethylene had no stimulating effect on the growth of hypocotyls or roots. The expression of each of the five genes detected in intact hypocotyls was inducible with auxin. The expression of auxin-inducible VR-ACS1 and VR-ACS6 was negatively regulated in the presence of auxin action and transport inhibitors. However, the transcripts of VR-ACS7, VR-ACO1, and VR-ACO2 were rarely influenced by the treatment of these inhibitors. These results suggest that additional growth factor(s), besides auxin, was (were) involved in the regulation of endogenous ethylene synthesis in etiolated mung bean seedlings.

The effect of reactive oxygen species on ethylene production induced by osmotic stress in etiolated mungbean seedling

After 10 h osmotic stress in 25% polyethylene glycol (PEG6000) solution (−1.8 MPa) at 25 °C in darkness, the etiolated mungbean seedlings were transferred to pure water for recovery. The ethylene release rate and the level of reactive oxygen species (ROS), including superoxide radical (O2−) and hydrogen peroxide (H2O2), were investigated during the recovery process. The results showed that ethylene production rate and amount of ROS increased dramatically after osmotic stress, and a close correlation was observed between ethylene release rate and concentrations of ROS. Inhibitors of ethylene biosynthesis, aminoethoxyvinylglycine (AVG) or aminooxyacetic acid (AOA), could reduce the ethylene release rate, but had no significant influence to the content of O2− and H2O2. As well as, silver thiosulfate (STS), an inhibitor of ethylene action, exhibited no obvious effect to the concentration of ROS, showing stress-inducible ethylene was not the cause for the increase of stress-inducible ROS. On the other hand, exogenous generator of superoxide radical (methylviologen, MV, or sodium dithionite, Na2S2O4) could enhance the ethylene production evidently, which could be inhibited by exogenous scavenger of superoxide radical (superoxide dismutase, SOD, or 1, 4-diazabicyclo (2,2,2) octane, DABCO). However, either exogenous H2O2 or catalase (CAT) had no significant influence on ethylene production. The results suggested that it was superoxide radical but not H2O2which was involved directly in osmotic stress-inducible ethylene biosynthesis. The dual-role of superoxide radical on stress ethylene biosynthesis was also discussed.

Proton pumping inorganic pyrophosphatase of endoplasmic reticulum-enriched vesicles from etiolated mung bean seedlings

Endoplasmic reticulum (ER)-enriched vesicles from etiolated hypocotyls of mung bean seedlings (Vigna radiata) were successfully isolated using Ficoll gradient and two-phase (polyethylene glycol-dextran) partition. The ER-enriched vesicles contained inorganic pyrophosphate (PPi) hydrolysis and its associated proton translocating activities. Antiserum prepared against vacuolar H+-pyrophosphatase (V-PPase, EC 3.6.1.1) did not inhibit this novel pyrophosphatase-dependent proton translocation, excluding the possible contamination of tonoplast vesicles in the ER-enriched membrane preparation. The optimal ratios of Mg2+/PPi (inorganic pyrophosphate) for enzymatic activity and PPi-dependent proton translocation of ER-enriched vesicles were higher than those of vacuolar membranes. The PPi-dependent proton translocation of ER-enriched vesicles absolutely required the presence of monovalent cations with preference for K+, but could be inhibited by a common PPase inhibitor, F-. Furthermore, ER H+-pyrophosphatase exhibited some similarities and differences to vacuolar H+-PPases in cofactor/substrate ratios, pH profile, and concentration dependence of F-, imidodiphosphate (a PPi analogue), and various chemical modifiers. These results suggest that ER-enriched vesicles contain a novel type of proton-translocating PPase distinct from that of tonoplast from higher plants.

Effects of Osmotic Stress on the Growth of Etiolated Mung Bean Seedlings

AbstractThe alterations in growth and water status of dark‐grown mung bean tissues following sudden osmotic stress were investigated. Photographic measurements of hypocotyl elongation revealed that stress (−0.2 to −0.8 MPa) induced growth cessation within one minute, with resumption occurring at lower rates after a latent period; periods which tended to increase in accordance with whether the seedlings were 4, 5 or 6 days old. Tissue water status was related to growth as water and osmotic potentials were highest in the expanded root region and the lowest in the growing hypocotyl region. These rankings were not verified under osmotic stress, and values were reduced in the course of 24 h. Turgor levels were similar in all tissues and were not altered by stress. The responses were not caused by substantial water loss, which was detected only after 24 h of stress, and could have resulted from the accumulation of osmotic solutes. The results indicate that plants exposed to sudden osmotic stress, besides exhibiting rapid growth cessation, show growth resumption at lower rates and no subsequent short‐term changes in any water status measure of the growing region. Because growth resumes without further detectable turgor increments, water status alterations are unlikely to be the cause of the observed immediate growth stoppage.

A complex containing both trypsin inhibitor and dehydroascorbate reductase activities isolated from mitochondria of etiolated mung bean ( Vigna radiata L. (Wilczek) cv. Tainan No. 5) seedlings

A complex containing trypsin inhibitor (TI) activity was extracted with 0.1 M TRIS buffer (pH 7.9) from trypsin‐treated mitochondria of etiolated mung bean seedlings, and further purified with a Superdex 200 FPLC column. This partially purified complex with an Mr about 820 kDa exhibited additional dehydroascorbate (DHA) reductase activity with specific activities of 0.21, 1.53 and 1.54 μmol ascorbate formed min −1 mg −1 protein at pH 6.0, 6.5 and 7.0, respectively, when glutathione was added. Much lower DHA reductase activity (0.013 and 0.026 μmol ascorbate formed min −1 mg −1 protein at pH 6.5 and 7.0, respectively) was found when glutathione was omitted. The isolated complex gave positive results when it was tested by TI activity staining after SDS‐PAGE, and could be recognized by a polyclonal antibody which was raised against 38 kDa sweet potato Kunitz‐type TI, one of the root storage proteins of sweet potato. The possible physiological functions of this complex with both TI and DHA reductase activities were discussed.

A complex containing both trypsin inhibitor and dehydroascorbate reductase activities isolated from mitochondria of etiolated mung bean (Vigna radiata L. (Wilczek) cv. Tainan No. 5) seedlings

A complex containing trypsin inhibitor (TI) activity was extracted with 0.1 M TRIS buffer (pH 7.9) from trypsin-treated mitochondria of etiolated mung bean seedlings, and further purified with a Superdex 200 FPLC column. This partially purified complex with an M(r) about 820 kDa exhibited additional dehydroascorbate (DHA) reductase activity with specific activities of 0.21, 1.53 and 1.54 mumol ascorbate formed min-1 mg-1 protein at pH 6.0, 6.5 and 7.0, respectively, when glutathione was added. Much lower DHA reductase activity (0.013 and 0.026 mumol ascorbate formed min-1 mg-1 protein at pH 6.5 and 7.0, respectively) was found when glutathione was omitted. The isolated complex gave positive results when it was tested by TI activity staining after SDS-PAGE, and could be recognized by a polyclonal antibody which was raised against 38 kDa sweet potato Kunitz-type TI, one of the root storage proteins of sweet potato. The possible physiological functions of this complex with both TI and DHA reductase activities were discussed.

Radiation inactivation analysis of H +-pyrophosphatase from submitochondrial particles of etiolated mung bean seedlings

Radiation inactivation analysis was employed to determine the functional masses of enzymatic activity and proton translocation of H +-pyrophosphatase from submitochondrial particles of etiolated mung bean seedlings. The activities of H +-pyrophosphatase decayed as a simple exponential function with respect to radiation dosage. D 37 values of 6.9±0.3 and 7.5±0.5 Mrad were obtained for pyrophosphate hydrolysis and its associated proton translocation, yielding molecular masses of 170±7 and 156±11 kDa, respectively. In the presence of valinomycin and 50 mM KCl, the functional size of H +-pyrophosphatase of tonoplast was decreased, while that of submitochondrial particles remained the same, indicating that they are two distinct types of proton pump using PP i as their energy source.

Molecular identification of cytokinin-specific binding protein

Synthetic phenylurea derivatives such as N-phenyl-N'-(4-pyridyl)urea (4PU) and N-(2-chloro-4-pyridyl)-N'-phenylurea (4PU30) have strong cytokinin activities. Using tritiated 4PU30 as a probe, we found the presence of a cytokinin-specific binding protein (CSBP) with high affinity for 4PU30 (Ka for 4PU30 = 4 x 10(10) M-1) in the soluble fraction of etiolated mung bean seedlings. We purified CSBP by the use of 4PU-Sepharose 4B, an affinity gel ligated with 4PU. Analysis of its cDNA revealed that CSBP was a novel member of a major pollen allergen/pathogenesis-related protein family with a calculated molecular weight of 17 kDa. Recombinant CSBP was expressed in Escherichia coli was confirmed to bind specifically to cytokinins.

Purification and cDNA cloning of cytokinin-specific binding protein from mung bean (Vigna radiata).

Synthetic urea derivatives such as N-phenyl-N'-(4-pyridyl)urea (4PU) and N-(2-chloro-4-pyridyl)-N'-phenylurea (4PU30) have strong cytokinin activities. Using tritiated 4PU30 as a probe, we previously established the presence of a cytokinin-specific binding protein (CSBP) of high affinity (Ka for 4PU30 = 4x10(10) M(-1)) in the soluble fraction of etiolated mung bean seedlings [Nagata, R., Kawachi, E., Hashimoto, Y. & Shudo, K. (1993) Biochem. Biophys. Res. Commun. 191, 543-549]. In this report, we purified CSBP by the use of 4PU-Sepharose 4B, an affinity gel liganded with 4PU. We determined partial amino acid sequences of CSBP and isolated its cDNA by reverse-transcription (RT) PCR. The cDNA encoded a protein with a calculated molecular mass of 17 kDa. A data base homology search revealed that CSBP is a novel member of a major pollen allergen/pathogenesis-related protein family. Recombinant CSBP was expressed in Escherichia coli and was confirmed to bind specifically to cytokinins.

High-pressure effects on vacuolar H+-ATPase from etiolated mung bean seedlings.

A high-hydrostatic-pressure technique was employed to study the structure-function relationship of plant vacuolar H+-ATPase from etiolated mung bean seedlings (Vigna radiata L.). When isolated vacuolar H+-ATPase was subjected to hydrostatic pressure, the activity of ATP hydrolysis was markedly inhibited in a time-, protein concentration- and pressure-dependent manner. The pressure treatment decreased both Vmax and Km of solubilized vacuolar H+-ATPase, implying an increase in ATP binding affinity, but a decrease in the ATP hydrolysis activity. Physiological substrate, Mg2+-ATP, augmented the loss of enzymatic activity upon pressure treatment. However, ADP, AMP, and Pi exerted substantial protective effects against pressurization. Steady-state ATP hydrolysis was more sensitive to pressurization than single-site ATPase activity. The inactivation of solubilized vacuolar H+-ATPase by pressure may result from changes in protein-protein interaction. The conformational change of solubilized vacuolar H+-ATPase induced by hydrostatic pressure was further determined by spectroscopic techniques. The inhibition of vacuolar H+-ATPase under pressurization involved at least two steps. Taken together, our work indicates that subunit-subunit interaction is crucial for the integrity and the function of plant vacuolar H+-ATPase. It is also suggested that the assembly of the vacuolar H+-ATPase complex is probably not random, but follows a sequestered pathway.

Involvement of tyrosine residue in the inhibition of plant vacuolar H +-pyrophosphatase by tetranitromethane

Plant vacuolar vesicles contain a novel H +-translocating pyrophosphatase (H +-PPase, EC 3.6.1.1). Modification of tonoplast vesicles and purified vacuolar H +-PPase from etiolated mung bean seedlings with tetranitromethane (TNM) resulted in a progressive decline in H +-translocating pyrophosphatase activity. The half-maximal inhibition was brought about by 0.6, 1.0, and 0.8 mM TNM for purified and membrane-bound H +-PPases, and its associated proton translocation, respectively. The maximal inhibition of vacuolar H +-PPase by TNM occurred at a pH value above 8. Loss of activity of purified H +-pyrophosphatase followed pseudo-first order rate kinetics, yielding a first-order rate constant ( k 2) of 0.039 s −1 and a steady-state dissociation constant of inactivation ( K i) of 0.02 mM. Covalent modification of vacuolar H +-PPase by TNM increased K m value of the enzyme for its substrate without a significant effect on V max. Double logarithmic plots of the pseudo-first order rate constant ( k obs) versus TNM concentration exhibited a slope of 0.88, suggesting that at least one tyrosine residue was involved in the inactivation of H +-PPase enzymatic activity. Further spectrophotometric measurements of the nitrated H +-pyrophosphatase indicated that TNM could modify approximately two tyrosine residues/subunit of the enzyme. However, Tsou's analysis revealed that only one of those modified tyrosine residues directly participated in the inhibition of enzymatic activity of vacuolar H +-PPase. The physiological substrate, i.e., dimagnesium pyrophosphate, provided substantial protection against inactivation by TNM. Moreover, NEM pretreatment of the enzyme decreased the number of subsequent nitration of vacuolar H +-PPase. Taken together, we suggest that vacuolar H +-pyrophosphatase contains a substrate-protectable tyrosine residue conferring to the inhibition of its activity and this tyrosine residue may be located in a domain sensitive to the modification of Cys-634 by NEM.

Purification and Characterization of a Cytochrome P450 (<italic>trans</italic>-Cinnamic Acid 4-Hydroxylase) from Etiolated Mung Bean Seedlings

In order to investigate biochemistry and molecular biology of P450 involvement in phenylpropanoid metabolism, we purified a P450 with trans-cinnamate 4-hydroxylase activity from microsomes of etiolated mung bean seedlings to electrophoretic homogeneity

Purification, characterization, and cDNA cloning of an NADPH-cytochrome P450 reductase from mung bean.

We report here the isolation and deduced amino acid sequence of the flavoprotein, NADPH-cytochrome P450 (cytochrome c) reductase (EC 1.6.2.4), associated with the microsomal fraction of etiolated mung bean seedlings (Vigna radiata var. Berken). An 1150-fold purification of the plant reductase was achieved, and SDS/PAGE showed a predominant protein band with an apparent molecular mass of approximately 82 kDa. The purified plant NADPH-P450 reductase gave a positive reaction as a glycoprotein, exhibited a typical flavoprotein visible absorbance spectrum, and contained almost equimolar quantities of FAD and FMN per mole of enzyme. Specific antibodies revealed the presence of unique epitopes distinguishing the plant and mammalian flavoproteins as demonstrated by Western blot analyses and inhibition studies. Peptide fragments from the purified plant NADPH-P450 reductase were sequenced, and degenerate primers were used in PCR amplification reactions. Overlapping cDNA clones were sequenced, and the deduced amino acid sequence of the mung bean NADPH-P450 reductase was compared with equivalent enzymes from mammalian species. Although common flavin and NADPH-binding sites are recognizable, there is only approximately 38% amino acid sequence identity. Surprisingly, the purified mung bean NADPH-P450 reductase can substitute for purified rat NADPH-P450 reductase in the reconstitution of the mammalian P450-catalyzed 17 alpha-hydroxylation of pregnenolone or progesterone.

Cytokinin-Specific Binding-Protein in Etiolated Mung Bean Seedlings

We have found that a synthetic urea derivative, N-(2-chloro-4-pyridyl)- N′-phenylurea (4PU30, forchlorfenuron), has even higher cytokinin activity than N 6-benzyladenine (BA). Using [ 3H]PU30 (2.83 TBq/mmol) as a probe, we confirmed chromatographically the presence of a high-affinity cytokinin-specific binding protein (CSBP) in whole-cell extract from etiolated mung bean seedlings. The apparent molecular weight of CSBP was estimated to be 21 kDa. The association constants (K a′s) of CSBP for 4PU30 and BA were calculated to be 4 × 10 10) M −1 and 3 × 10 9 M −1, respectively. Various active cytokinins showed mutually competitive binding to CSBP, and their affinities for CSBP corresponded well to their cytokinin activities at the tissue level.

Inhibition of tonoplast ATPase from etiolated mung bean seedlings by fluorescein 5'-isothiocyanate.

Fluorescein 5'-isothiocyanate (FITC) was used to modify the lysine residue in the active site of tonoplast H(+)-ATPase from etiolated mung-bean (Vigna radiata L.) seedlings. FITC caused marked inactivation of the enzyme activities of both membrane-bound and soluble ATPase and its associated H+ translocation. The SDS/PAGE pattern revealed that the FITC-binding site was in the large (A) subunit of ATPase. Inhibition could be substantially prevented by its physiological substrate ATP, pyrophosphate and nucleotides in the decreasing order: ATP greater than pyrophosphate greater than ADP greater than AMP greater than GTP greater than CTP greater than UTP. The mode of inhibition by FITC was competitive with respect to ATP. Loss of ATPase activity followed pseudo-first-order kinetics with a Ki of 0.33 mM, a minimum inactivation half-time of 110 s, and a first-order rate constant of 0.244 s-1. A double-logarithmic plot of apparent rate constant versus FITC concentration gave a slope of 0.913, indicating that inactivation results from reaction of at least one lysine residue at the catalytic site of the large subunit. Labelling studies indicated that the incorporation of approx. 1 mol of FITC/mol of ATPase is sufficient to inhibit ATPase completely. The enhancement and blue shift of emission maxima of FITC after modification of ATPase indicated that the labelled lysine residue was located in a relatively hydrophobic domain.

Interaction between Auxin–binding Protein–I and RNA Polymerase II

Interactions between auxin–binding protein–I (ABP–I), purified from etiolated mung bean seedlings, and nuclear components from mung bean tissues were investigated. When NaCI–solubilized components of chromatin were put on an affinity column of ABP–I–Iinked Sepharose 4B, a small amount of the material was retained on the affinity column and was eluted with 1 M NaCl. RNA polymerase II activity was detected in the eluted fraction. Partially purified RNA polymerase II from mung bean nuclei and purified RNA polymerase II from wheat germ also bound to ABP–I. Indole–3–acetic acid was not necessary for the binding of RNA polymerase II to ABP–I. Acid–denatured ABP–I did not bind to RNA polymerase II from wheat germ. The addition of ABP–I to the reaction mixture for RNA synthesis in vitro caused a stimulation of the activity of wheat germ RNA polymerase.

Purification of a Soluble Cytockinin-binding Protein from Etiolated Mung Bean Seedlings.

A cytokinin-binding protein (CBP) was purified from a crude extract of etiolated mung bean seedlings by a protocol involving affinity chromatography on benzyladenine-linked Sepharose 4B, ion exchange chromatography on DEAE-Sephadex A50, and gel filtration on Sphacryl S-400. The molecular weight was estimatd to be about 200,000 by gel filtration. CBP appeared as two bands corresponding to molecular weights of about 45,000 and 48,000 on SDS-polyacrylamide gel electrophoresis. The dissociation constant for benzyladenine was 7.5 x 10(-7) M. (14)C-Benzyladenine-binding to CBP was reversible and could be inhibited by the addition of kinetin or trans-zeatin. Adenine, AMP, and ADP had no inhibitory effect on the binding of (14)C-benzyladenine to CBP but the addition of ATP to the assay mixture enhanced the binding.