Isolation and characterization of a cytokinin-binding protein from cucumber cotyledons

A protein which binds specifically to [³H]-zeatin has been isolated from cucumber cotyledons by chromatographic techniques. Its binding to [³H]-zeatin was inhibited remarkably by the addition of non-radioactive cytokinins and the order of inhibition was zeatin > -zeatin riboside > N⁶-(²-isopentenyl)adenine > N⁶-(²-isopentenyl)adenosine > N⁶-benzyl-adenosine > kinetin riboside. This protein behaved as a soluble protein with an apparent molecular size of 43,000 daltons on gel filtration through calibrated Sephadex G-100 column. The dissociation constant, K_d, of the protein-zeatin complex was about 4 × 10^–7 M.     

Cytokinins in Azotobacter vinelandii Culture Medium

Azotobacter vinelandii OP was grown to stationary phase in defined medium. The cell-free culture medium was analyzed for cytokinin content by XAD-2 and Sephadex LH-20 chromatography, thin-layer chromatography, tobacco callus bioassay, and enzyme immunoassay. Three cytokinin-active fractions were detected and tentatively identified as trans-zeatin, isopentenyladenosine, and isopentenyladenine. The total cytokinin activity was equivalent to 0.75 μg of kinetin per liter.     

Kinetin incorporated into tobacco callus ribosomal RNA and transfer RNA preparations

Kinetin, N⁶-furfuryladenine, was incorporated into tobacco (nicotiana tabacum L., var. Wis. No. 38) callus RNA isolated from rapidly growing tissue cultured in the presence of N⁶-furfuryladenine-8-¹⁴C or unlabeled kinetin. Approximately 0.7% of the radioactivity in the labeled kinetin added to the medium was recovered as N⁶-furfuryladenosine (fr⁶A) in the rRNA and tRNA preparations from the tobacco callus. The rRNA contained over 90% of these fr⁶ A moieties. The extent of kinetin incorporation was four times greater than that observed for N⁶-benzyladenine. The radiochemical purity of the recovered fr⁶ A was confirmed by three successive chromatographic purifications on Sephadex columns (LH-20 eluted with 35% ethanol, G-10 eluted with 20% ethanol, and LH-20 eluted with water). A cytokinin-active ribonucleoside with elution volumes corresponding to fr⁶ A was isolated from the tobacco callus rRNA preparation. This compound was analyzed by gas-liquid chromatography and rigorously characterized as N⁶-furfuryladenosine by gas-liquid chromatography-mass spectrometry of the trimethylsilyl derivative.     

Differential cytokinin structure-activity relationships in phaseolus

The activities of eight cytokinins in promoting callus growth were tested in two Phaseolus genotypes, P. vulgaris L. var. Great Northern, and P. lunatus L. var. Kingston. The structural feature which contributes to the major genotypic difference in cytokinin structure-activity relationships is the presence or absence of a double bond at the 2,3-position of the isoprenoid N⁶ side chain. In Kingston, trans-zeatin was 3-fold more active than dihydrozeatin and 30-fold more active than cis-zeatin. The activities of N⁶-(Δ²-isopentenyl)adenine and N⁶-isopentyladenine were nearly the same. In Great Northern, however, dihydrozeatin was at least 30-fold more active than both trans-zeatin and cis-zeatin, and N⁶-isopentyladenine was 100-fold more active than N⁶-(Δ²-isopentenyl)adenine. The results suggest the possibility of employing cytokinin structure-activity relationships in distinguishing genotypic differences in cytokinin function and metabolism.     

Cytokinins in shoots of the chestnut tree

Five cytokinins, trans-zeatin, 9-β-d-ribosyl-trans-zeatin, O-β-d-glucosyl-trans-zeatin, N⁶-(3-methyl-but-2-enyl)adenine and N⁶-(3-methyl-but-2-enyl)adenosine were identified in shoots of the chestnut tree.     

Cytokinin binding proteins from mammalian sera

A cytokinin-binding protein fraction was isolated from normal rabbit sera by affinity chromatography. The protein fraction bound tritium labelled N⁶- (δ2-risopentenyl) adenosine and the order of inhibition of this binding by competing non-radioactive compounds was, N⁶-(δ²-isopentenyl) adenosine < N⁶-benzyIadenosine < zeatin-riboside > N⁶-(δ²-isopentenyl) adenine < kinetin riboside > adenosine. The protein fraction showed broad specificity, the prefered cytokinin being N⁶-(δ²-isopentenyl) adenosine. This is the first report of the isolation of cytokinin binding proteins from mammalian sources.     

Cytokinins of sycamore spring sap

Summary The cytokinins present in the spring sap of Acer pseudoplatanus L. were investigated. Ribosyl-trans-zeatin, trans-zeatin and dihydrozeatin were isolated and identified by combined gas chromatography-mass spectrometry (GC-MS). A number of other cytokinin active fractions were observed. One of these was less polar than zeatin and did not behave as any known cytokinin. Two other fractions were more polar than ribosylzeatin and were unstable. A decomposition product of one of these was identified as ribosyl-trans-zeatin by GC-MS. The possible nature of the unstable compounds is discussed. Data on the changes in cytokinin activity of the various fractions during spring 1973 are presented and discussed.Abbreviations GLC gas-liquid chromatography - GG-MS combined gas chromatography-mass spectrometry - KE kinetin equivalents - TLC thin-layer chromatography - TMS trimethylsilyl - tRNA transfer RNA - i⁶ Ade 6-(3-methylbut-2-enylamino)-purine - i⁶ Ado 6-(3-methylbut-2-enylamino)-9--D-ribofuranosyl-purine     

An enzyme mediating the conversion of zeatin to dihydrozeatin in phaseolus embryos

A reductase catalyzing the conversion of zeatin to dihydrozeatin was detected in soluble fractions of immature Phaseolus vulgaris embryos. The enzyme was partially purified by ammonium sulfate fractionation and affinity, gel filtration, and anion exchange chromatography. NADPH was the only cofactor required for enzyme activity, and the pH optimum was 7.5 to 8.0. The enzyme did not recognize compounds closely related to zeatin, such as ribosylzeatin, cls-zeatin, O-xylosylzeatin, N⁶-(Δ²-isopentenyl)adenine, or N⁶-(Δ²-isopentenyl)adenosine. No conversion of dihydrozeatin to zeatin by the enzyme was observed. Two forms of the reductase could be separated by either gel filtration or anion exchange high performance liquid chromatography. The high molecular weight isozyme (M_r 55,000 ± 5,000) eluted as the second peak from the anion exchange column, while the low molecular weight isozyme (M_r 25,000± 5000) was less negatively charged. The results suggest that side chain reduction occurs at the free base level. In addition, Phaseolus embryos are useful for the detection of zeatin-specific metabolic enzymes.     

Ligand Specificity of a High Affinity Cytokinin-binding Protein

A soluble cytokinin-binding protein from wheat germ that has a high affinity for a range of purine cytokinins also interacts with a variety of nonpurine compounds that can affect cytokinin-modified processes in animal or plant cells or which bind to proteins known to interact with certain cytokinins. A variety of structurally disparate compounds which inhibit chloroplast photosystem II activity (including phenylurea, carbanilate, and alkylamino-2-chloro-sym-triazine compounds) displace kinetin from the protein in an apparently competitive fashion. However, various energy transfer inhibitors (including organotin compounds and N,N′-dicy-clohexylcarbodiimide) also inhibit kinetin binding to the protein. N⁶,2-0′-Dibutyryl-3′,5′-cyclic AMP and 1-methyl-3-isobutylxanthine (the effects of which on fibroblast morphology and motility can be mimicked by cytokinins) are inhibitors of kinetin binding to the protein. A variety of compounds that can have antimitotic effects (including 1-methyl-3-isobutylxanthine and certain alkylated cyclic nucleotide, carbanilate, and tryptamine compounds) displace kinetin from the protein. However, a variety of indole derivatives also displace kinetin from the cytokinin-binding protein, which in a qualitative sense has a broad ligand specificity.     

Production and characterization of antibodies and establishment of a radioimmunoassay for ribosylzeatin

An antibody directed towards ribosyl-trans-zeatin has been produced and characterized. The antiserum was produced in rabbits using ribosyl-zeatin-bovine serum albumin as an immunogen. A radioimmunoassay which employed this antiserum and a tritiated antigen was established. As little as 10 picomoles ribosyl-trans-zeatin could be detected. The specificity of the antiserum was measured in the radioimmunoassay by using nonradioactive nucleosides as competitive inhibitors. Changes in position N⁶ were more effective in decreasing antibody recognition than changes in position 2. Of particular interest was the interaction of the isomer ribosyl-cis-zeatin. This compound was significantly less active as an inhibitor than ribosyl-trans-zeatin, demonstrating that the antibody was sensitive to minor changes in the structure of the antigen.     

Purification and characterization of 1-aminocyclopropane-1-carboxylate N-malonyltransferase from etiolated mung bean hypocotyls

1-Aminocyclopropane-1-carboxylate (ACC) N-malonyltransferase converts ACC, an immediate precursor of ethylene, to the presumably inactive product malonyl-ACC (MACC). This enzyme plays a role in ethylene production by reducing the level of free ACC in plant tissue. In this study, ACC N-malonyltransferase was purified 3660-fold from etiolated mung bean (Vigna radiata) hypocotyls, with a 6% overall recovery. The final specific activity was about 83,000 nmol of MACC formed mg⁻¹ protein h⁻¹. The five-step purification protocol consisted of polyethylene glycol fractionation, Cibacron blue 3GA-agarose chromatography using salt gradient elution, Sephadex G-100 gel filtration, MonoQ anion-exchange chromatography, and Cibacron blue 3GA-agarose chromatography using malonyl-CoA plus ACC for elution. The molecular mass of the native enzyme determined by Sephadex G-100 chromatography was 50 ± 3 kD. Protein from the final purification step showed one major band at 55 kD after sodium dodecyl sulfate polyacrylamide gel electrophoresis, indicating that ACC N-malonyltransferase is a monomer. The mung bean ACC N-malonyltransferase has a pH optimum of 8.0, an apparent K_m of 0.5 mm for ACC and 0.2 mm for malonyl-coenzyme A, and an Arrhenius activation energy of 70.29 kJ mol⁻¹ degree⁻¹.     

Incorporation of 3H-adenine into free cytokinins by cytokinin-autonomous tobacco callus tissue

Cytokinin-autonomous tobacco callus was incubated in defined mineral medium containing ³H-adenine for 60 minutes. Radioactivity was incorporated into the four predominant free cytokinins, ribosyl-$\text{trans}$-zeatin, $\text{trans}$-zeatin, N⁶-(Δ²-isopentenyl) adenosine and N⁶-(Δ²-isopentenyl) adenine. The bases were more abundant than their respective ribosides, N⁶-(Δ²-isopentenyl) adenine being the most abundant cytokinin. No discrete peaks of radioactivity could be detected on the HPLC column eluate corresponding to the elution volumes of $\text{cis}$-zeatin and ribosyl-$\text{cis}$-zeatin.     

Cytokinins in tobacco crown gall tumors

Bacteria-free tobacco ($\text{Nicotiana tabacum}$ cv. Wisconsin #38) crown gall strains incited by $\text{Agrobacterium tumefaciens}$ C58, 27, B6, CGIC, and AT4 have been analyzed for cytokinin content with the tobacco callus bioassay. All tumor strains contained high total levels of cytokinins ranging from 4–810 kinetin equivalents per kg fresh weight compared to 0.5 kinetin equivalents per kg for normal callus growing on medium with 0.1 μM N6-benzyladenine. Fractionation on a column of Sephadex LH-20 separated cytokinin activity from B6 tumors into a number of components among which ribosyl-$\text{trans}$-zeatin has been purified and characterized based on uv spectrum, biological activity and mass spectrum.     

Studies on purification of a lectin from fruiting bodies of the edible shiitake mushroom Lentinus edodes

A lectin, with a specificity for N-acetylgalactosamine and N-acetylglucosamine and a molecular weight of 43 kDa, was isolated from fruiting bodies of the edible shiitake mushroom Lentinus edodes. The purification procedure entailed extraction with aqueous buffer, ammonium sulfate precipitation, gel filtration on Sephadex G-100 and affinity chromatography on N-acetylgalactosamine-agarose. The lectin was unique in that it was tenaciously bound on anion exchangers including DEAE-cellulose, DEAE-Sepharose, DEAE-Sephadex, Q-Sepharose, Dowex and PEI-cellulose and also on hydroxyapatite and phenyl Sepharose. It was largely unadsorbed on cation exchangers including CM-cellulose, CM-Sepharose, SP-Sepharose and Amberlite, and also on protein G-Sepharose, Red Sepharose and Affi-gel Blue gel, wheat germ lectin-Sepharose and p-aminophenyl-d-glucopyranoside agarose.     

Immunoaffinity co-purification of cytokinins and analysis by high-performance liquid chromatography with ultraviolet-spectrum detection

A rapid methodology for the simultaneous analysis of a large number of cytokinins is presented. The cross-reactivity of a mixture of polyclonal antibodies against zeatin riboside and isopentenyladenosine was exploited in a protocol that can be used for immunoaffinity purification of 23 additional cytokinins. Ligands include the cytokinin bases zeatin, dihydrozeatin, isopentenyladenine, benzyl-adenine and kinetin, and their corresponding nucleoside, nucleoside-5′-monophosphate, and 9-glucoside derivatives, as well as cis-zeatin, cis-zeatin riboside, the 2-methylthiol derivatives of isopentenyladenosine and zeatin riboside, and benzyl-adenine-3-glucoside. Mixtures of cytokinins could be retained with high recoveries of all the components. Immunoaffinity purification of extracts of Arabidopsis thaliana (L.) Heynh. and Solarium tuberosum L. gave fractions clean enough, as verified by gas chromatographymass spectrometry (GC-MS), to allow analysis of endogenous cytokinins using a single high-performance liquid chromatography (HPLC) step with on-line UV-spectrum detection. The detection limit was 4–6 pmol. The procedure described forms a routine assaying technique that is faster and simpler, yet yields better qualitative and quantitative information than the commonly used procedure of immunoassaying of HPLC fractions.     

Kinetin inhibition of h-uracil and C-leucine incorporation by tobacco cells in suspension culture

The rate of ¹⁴C-leucine and ³H-uracil incorporation by tobacco cells (Nicotiana tabaccum var. Samsun N.N.) in suspension culture was simultaneously decreased by the addition of kinetin at concentrations above 2.5 × 10⁻⁵m. Ribosomal RNA was the first RNA species affected by kinetin. The purine derivatives, adenine and N⁶-methyl-aminopurine, which exhibit low cytokinin activity overcame the inhibitory effects of kinetin. However, purine derivatives without cytokinin activity, guanine, N^6,6-dimethyl-aminopurine, and 2-aminopurine, did not relieve kinetin inhibition.     

Qualitative and semi-quantitative analyses of cytokinins using LC/APCI-MS in combination with ELISA

Application of liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (LC/APCI-MS) for the analysis of cytokinins was examined. The fragmentation of cytokinins was studied using authentic trans-zeatin (t-Z), trans-zeatin riboside (t-ZR), isopentenyl adenine (i⁶Ade), isopentenyl adenosine (i⁶Ado), benzyl adenine (BAde), benzyl adenosine (BAdo), and kinetin. These cytokinins were effectively ionized by APCI in aqueous acetonitrile. t-Z, i⁶Ade, BAde, and kinetin showed prominent quasi-molecular ions of [M + H]⁺, and ribosylcytokinins clearly showed both [M + H]⁺ and a characteristic fragment ion ([M + H-ribose]⁺), giving some information about their structures. The qualitative and semi-quantitative analyses of cytokinins by LC/APCI-MS were validated in combination with enzyme-linked immunosorbent assay (ELISA) through the analysis of t-ZR in the teratoma of Nicotiana tobacum. t-ZR was conclusively identified and a semi-quantitative estimate of its endogenous levels were provided by the combination of LC/APCI-MS and ELISA. The quantified values obtained by LC/ APCI-MS (single ion detection) and ELISA are in close agreement.