共查询到20条相似文献,搜索用时 78 毫秒
1.
Kinetic comparison of cytokinin nucleosidase activity isolated from normally ripening and mutant tomato varieties 下载免费PDF全文
Kinetic parameters for cytokinin nucleosidase activity which catalyzes the deribosylation of N6(Δ2-isopentenyl)adenosine (I6Ado) to produce the more “active” free base N5(Δ2-isopenetyl)adenine (I6Ade) were compared for a normally ripening tomato (Lycopersicon esculentum L.) cultivar Rutgers, and two mutant tomato varieties (Nor and Rin). Km for nucleosidase activity in Rutgers was lower (Km = 0.1 millimolar) than that in either Nor (Km = 0.14 millimolar) or Rin (Km = 0.13 millimolar). 相似文献
2.
Metabolism of cytokinin: ribosylation of cytokinin bases by adenosine phosphorylase from wheat germ 下载免费PDF全文
As part of the study of cytokinin metabolic pathways, an enzyme, adenosine phosphorylase (EC 2.4.2.-), which catalyzed the ribosylation of N6-(Δ2-isopentenyl)adenine, N6-furfuryladenine, and adenine to form the corresponding nucleosides, was partially purified from wheat (Triticum aestivum) germ. The pH optimum for the ribosylation of the cytokinins and adenine was from 6.5 to 7.8; for guanine and hypoxanthine it was from 7.0 to 8.5 At pH 7.2 (63 millimolar N-2-hydroxyethyl piperazine-N′-ethanesulfonic acid) and 37 C the Km for N6-(Δ2-isopentenyl)adenine was 57.1 micromolar; N6-furfuryladenine, 46.5 micromolar; adenine, 32.2 micromolar; and the Vmax for N6-(Δ2-isopentenyl)adenine, N6-furfuryladenine, and adenine were 134.7, 137.1, and 193.1 nanomoles per milligram protein per minute, respectively. The equilibrium constants of the phosphorolysis of N6-(Δ2-isopentenyl)adenosine and adenosine by this enzyme indicated that the reaction strongly favored nucleoside formation. This enzyme was shown to be distinct from inosine-guanosine phosphorylase based on the differences in the Sephadex G-100 gel filtration behaviors, pH optima, and the product and p-hydroxymercuribenzoate inhibitor studies. These results suggest that adenosine phosphorylase may play a significant role in the regulation of cytokinin metabolism. 相似文献
3.
As part of the study of the possible role(s) of CBF-1, a cytokinin-binding protein abundant in wheat embryo, a cytokinin oxidase was found in wheat (Triticum aestivum L.) germ and partially purified by conventional purification techniques and high performance chromatofocusing. This preparation catalyzes conversion of N6-(Δ2-isopentenyl)adenosine to adenosine at a Vmax of 0.4 nanomol per milligram protein per minute at 30°C and pH 7.5, the Km being 0.3 micromolar. This high affinity and the apparent molecular weight of 40,000 estimated by high performance gel permeation on a Spherogel TSK-3000 SW column indicate that this enzyme is different from other cytokinin oxidases previously reported. Oxygen is required for the reaction, as for other cytokinin oxidases already described. N6-(Δ2-isopentenyl)adenine and zeatin riboside are also degraded, but N6-(Δ2-isopentenyl)adenosine-5′-monophosphate is apparently not a substrate. Benzyladenine is degraded, but to a small extent, and it inhibits slightly the degradation of N6-(Δ2-isopentenyl)adenosine. The degradation of N6-(Δ2-isopentenyl)adenosine is strongly inhibited by diphenylurea and its highly active derivative N-(2-chloro-4-pyridyl)-N′-phenylurea. 相似文献
4.
Kinetics of N-(Delta-Isopentenyl)Adenosine Degradation in Tobacco Cells: EVIDENCE OF A REGULATORY MECHANISM UNDER THE CONTROL OF CYTOKININS 下载免费PDF全文
Uptake and degradation of the cytokinin, N6-(Δ2-isopentenyl) adenosine, were studied in tobacco cells grown as cell suspensions. Degradation occurs by cleavage of the isopentenyl chain which gives adenylic products. Rate of N6(Δ2-isopentenyl)[8-14C]adenosine degradation increases several-fold after a 3- to 4-hour delay when cells have been exposed to a cytokinin. Consequently, only rates of N6-(Δ2-isopentenyl)adenosine degradation measured during the first 3 hours of incubation with [8-14C]-N-6(Δ2-isopentenyl)adenosine are representative of the intrinsic in vivo cytokinin degradative activity of tobacco cells. Within these limits, it appears that cytokinin degradative activity is high in cytokinin-autonomous tobacco cells, as indicated by the half life of the supplied N6(Δ2 isopentenyl adenosine (about 3 hours) when it is supplied at the physiological concentration of 0.2 micromolar. This cytokinin degradative activity appears to be under the control of cytokinins themselves because N6-(Δ2-isopentenyl)adenosine degradative activity is increased several-fold following a 3- to 4-hour delay after these cells have been exposed to a cytokinin. 相似文献
5.
Cytokinins: Metabolism and Biological Activity of N-(Delta-Isopentenyl)adenosine and N-(Delta-Isopentenyl)adenine in Tobacco Cells and Callus 下载免费PDF全文
The cytokinin, N6-(Δ2-isopentenyl)adenine, is found to be at least 3.3 times as active as N6-(Δ2-isopentenyl)adenosine in promoting the growth of cytokinin-requiring tobacco (Nicotiana tabacum) callus. Absorption rates of N6-(Δ2-isopentenyl)adenine and N6-(Δ2-isopentenyl)adenosine by tobacco cells in liquid suspension do not differ significantly. In these cells, N6-(Δ2-isopentenyl)adenosine-5′-monophosphate, di-, and triphosphate are synthesized in both cases, but 7-glucosylation occurs significantly only with N6-(Δ2-isopentenyl)adenine, protecting thereby its N6-isopentenyl side chain from cleavage. Degradation by N6-side chain removal appears to be intense, leading to the formation of adenine, adenosine, and adenylic nucleotides. Thus, it is suggested that N6-(Δ2-isopentenyl)adenine-7-glucoside is a protected or storage form of the cytokinin which could account for the higher biological activity of N6-(Δ2-isopentenyl)adenine than of N6-(Δ2-isopentenyl)adenosine. 相似文献
6.
Metabolism of Cytokinin : DEPHOSPHORYLATION OF CYTOKININ RIBONUCLEOTIDE BY 5'-NUCLEOTIDASES FROM WHEAT GERM CYTOSOL 下载免费PDF全文
Two forms (F-I and F-II) of 5′-nucleotidases (5′-ribonucleotide phosphohydrolase, EC 3.1.3.5) which catalyze the dephosphorylation of N6-(Δ2-isopentenyl)adenosine 5′-monophosphate and AMP to form the corresponding nucleosides were partially purified from the cytosol of wheat (Triticum aestivum) germ. Both the F-I (molecular weight, 57,000) and F-II (molecular weight, 110,000) 5′-nucleotidases dephosphorylate the ribonucleotides at an optimum pH of 7. The Km values for the cytokinin nucleotide are 3.5 micromolar (F-I enzyme) and 12.8 micromolar (F-II enzyme) in 100 millimolar Tris-maleate buffer (pH 7) at 37 C. The F-I enzyme is less rapidly inactivated by heating than is the F-II enzyme. Both nucleotidases hydrolyze purine ribonucleoside 5′-phosphates, AMP being the preferred substrate. N6-(Δ2-isopentenyl)Adenosine 5′-monophosphate is hydrolyzed at a rate 72 and 86% that of AMP by the F-I and F-II nucleotides, respectively. Phenylphosphate and 3′-AMP are not substrates for the enzymes. It is proposed that dephosphorylation of cytokinin nucleotide by cytosol 5′-nucleotidases may play an important role in regulating levels of “active cytokinin” in plant cells. 相似文献
7.
Localization of cytokinin biosynthetic sites in pea plants and carrot roots 总被引:6,自引:4,他引:2 下载免费PDF全文
The biosynthesis of cytokinins was examined in pea (Pisum sativum L.) plant organs and carrot (Daucus carota L.) root tissues. When pea roots, stems, and leaves were grown separately for three weeks on a culture medium containing [8-14C]adenine without an exogenous supply of cytokinin and auxin, radioactive cytokinins were synthesized by each of these organs. Incubation of carrot root cambium and noncambium tissues for three days in a liquid culture medium containing [8-14C]adenine without cytokinin demonstrates that radioactive cytokinins were synthesized in the cambium but not in the noncambium tissue preparation. The radioactive cytokinins extracted from each of these tissues were analyzed by Sephadex LH-20 columns, reverse phase high pressure liquid chromatography, paper chromatography in various solvent systems, and paper electrophoresis. The main species of cytokinins detectable by these methods are N6-(Δ2-isopentyl_adenine-5′-monophosphate, 6-(4-hydroxy-3-methyl-2-butenyl-amino)-9-β-ribofuranosylpurine-5′- monophosphate, N6-(Δ2-isopentenyl)adenosine, 6-(4-hydroxy-3-methyl-2-butenylamino)-9-β-ribofuranosylpurine, N6-(Δ2-isopentenyl)adenine, and 6-(4-hydroxy-3-methyl-2-butenylamino)purine. On the basis of the amounts of cytokinin synthesized per gram fresh tissues, these results indicate that the root is the major site, but not the only site, of cytokinin biosynthesis. Furthermore, cambium and possibly all actively dividing tissues are responsible for the synthesis of this group of plant hormones. 相似文献
8.
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. 相似文献
9.
Three cytokinin-over-producing mutants of the moss, Physcomitrella patens, have been shown to convert [8-14C]adenine to N6-[14C](Δ2-isopentenyl)adenine, the presence of which was confirmed by thin layer chromatography, high performance liquid chromatography, and recrystallization to constant specific radioactivity. The labeled cytokinin was detected in the culture medium within 6 hours and the tissue itself appears to contain both labeled N6-(Δ2-isopentenyl)adenine and N6-(Δ2-isopentenyl)adenosine monophosphate. 相似文献
10.
Adenosine kinase was partially purified from wheat germ. This enzyme preparation, which was devoid of adenine phosphoribosyltransferase and nearly free of adenosine deaminase but contained adenylate kinase, rapidly phosphorylated adenosine and a cytokinin, N6-(δ2-isopentenyl)adenosine. Electrophoretic analysis indicated that only N6-(δ2-isopentenyl)adenosine-monophosphate was formed from the cytokinin while about 55% AMP, 45% ADP, and a trace of ATP were formed from adenosine. The biosynthesized nucleoside monophosphates were quantitatively hydrolyzed to the corresponding nucleosides by 5′-nucleotidase and the isopentenyl side chain of the phosphorylated cytokinin was not cleaved. The enzyme did not catalyze phosphorylation of inosine. 相似文献
11.
Cytokinin structure-activity relationships and the metabolism of N-(delta-isopentenyl)adenosine-8-C in phaseolus callus tissues 下载免费PDF全文
The activities of the free base and ribonucleoside forms of cytokinins bearing saturated and unsaturated N6-isoprenoid side chains have been examined in callus cultures derived from Phaseolus vulgaris cv. Great Northern, P. lunatus cv. Kingston, and the interspecific hybrid Great Northern × Kingston. In callus of cv. Great Northern, cytokinins bearing saturated side chains (N6-isopentyladenine, N6-isopentyladenosine, dihydrozeatin, and ribosyldihydrozeatin) were always more active than the corresponding unsaturated analogs (N6-[Δ2-isopentenyl]adenine, N6-[Δ2-isopentenyl]adenosine, zeatin, and ribosylzeatin). In callus of cv. Kinston, the cytokinins bearing unsaturated side chains were either more active or equally as active as the saturated compounds. These differences in cytokinin structure-activity relationships were correlated with differences in the metabolism of 14C-N6-(Δ2-isopentenyl)adenosine. In Great Northern tissues, this cytokinin was rapidly degraded to adenosine; in Kingston tissues, the major metabolite was the corresponding nucleotide. The growth responses of callus of the interspecific hybrid were intermediate between the parental tissues, and the metabolism of 14C-N6-(Δ2-isopentenyl)adenosine by the hybrid callus exhibited characteristics of both parental tissues. The results are consistent with the hypothesis that the weak activity of cytokinins with unsaturated side chains in promoting the growth of Great Northern callus is due to the rapid conversion of these cytokinins to inactive metabolites. 相似文献
12.
Isolation and Estimation of Cytokinins and Cytokinin-Containing Transfer RNAs from Cucumis sativus L. var. Guntur Seedlings 下载免费PDF全文
N6-(Δ2-Isopentenyl) adenosine antibodies were used for the isolation of free cytokinins and cytokinin-containing tRNAs from parts of Cucumis sativus L. var. Guntur seedlings and for the estimation of cytokinins in them. Immobilized N6-(Δ2-isopentenyl) adenosine antibodies retained tRNAs containing N6-(Δ2-isopentenyl) adenosine and N6-(4-hydroxy-3-methylbut-2-enyl) adenosine with equal efficiencies. There were at least five cytokinins in the free form in cucumber seedlings. N6-(4-Hydroxy-3-methylbut-2-enyl) adenosine, N6-(Δ2-isopentenyl) adenosine, and N6-(Δ2-isopentenyl) adenine were present at least to the extent of 80, 23, and 9 nanograms, respectively, in the cotyledons and 40, 6, and 3 nanograms, respectively, in the decotyledonated seedlings per gram of tissue. Only two cytokinins were found in the tRNAs of cucumber cotyledons, namely N6-(Δ2-isopentenyl) adenosine and N6-(4-hydroxy-3-methylbut-2-enyl) adenosine in amounts of 12 and 318 nanograms, respectively, per gram of tissue. Immunoaffinity chromatographic analysis of radiolabeled aminoacyl tRNAs from cucumber cotyledons showed that tRNAPhe and tRNATyr contained cytokinins whereas tRNAAla did not. 相似文献
13.
Chong-Maw Chen Debra K. Melitz Fred W. Clough 《Archives of biochemistry and biophysics》1982,214(2):634-641
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). Km values for N6-(Δ2-isopentenyl)adenine, N6-furfuryladenine, N6-benzyladenine, and adenine are 130, 110, 154, and 74 μm, 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.” 相似文献
14.
Frankia sp. HFP ArI3 (host plant Alnus rubra Bong.) was grown in defined medium and the culture solution was analyzed for the presence of various cytokinins and related compounds. N6- (Δ2-isopentenyl) adenosine was the only cytokinin detected by both high performance liquid chromatography and gas chromatography-mass spectrometry, at levels of approximately 1 ng/ml culture medium. 相似文献
15.
trans-Ribosylzeatin: Its Biosynthesis in Zea mays Endosperm and the Mycorrhizal Fungus, Rhizopogon roseolus 下载免费PDF全文
When [8-14C]-N6-(Δ2-isopentenyl) adenosine is incubated with the endosperm of corn (2 weeks after pollination), it is converted to [14C]-N6-(4-hydroxy-3-methylbut-2-trans-enyl) adenosine, trans-ribosylzeatin. This biosynthetic step, N6-(Δ2-isopentenyl) adenosine to ribosylzeatin, also occurs in the mycorrhizal fungus, Rhizopogon roseolus. 相似文献
16.
Effects of Thidiazuron on Cytokinin Autonomy and the Metabolism of N-(Delta-Isopentenyl)[8-C]Adenosine in Callus Tissues of Phaseolus lunatus L 下载免费PDF全文
The effects of a highly cytokinin-active urea derivative, N-phenyl-N′-1,2,3-thiadiazol-5-ylurea (Thidiazuron), and zeatin on cytokinin-autonomous growth and the metabolism of N6-(Δ2-isopentenyl)[8-14C]adenosine ([14C]i6 Ado) were examined in callus tissues of two Phaseolus lunatus genotypes, cv Jackson Wonder and P.I. 260415. Tissues of cv Jackson Wonder maintained on any concentration of Thidiazuron became cytokinin autonomous, whereas only tissues exposed to suboptimal concentrations of zeatin displayed cytokinin-autonomous growth. Tissues of P.I. 260415 remained cytokinin dependent under all these conditions. The metabolism of [14C]i6 Ado was similar for the two genotypes, but differed with the medium used. [14C]i6 Ado was rapidly converted to N6-(Δ2-isopentenyl)[8-14C]adenosine 5′-P ([14C]i6 AMP) by tissues grown on zeatin-containing medium, whereas only traces of the nucleotide were formed in tissues grown on medium with Thidiazuron. Incubation with [14C] i6 AMP of tissues grown in the presence of Thidiazuron resulted in rapid conversion to [14C]i6 Ado, while [14C]i6 AMP persisted in tissues maintained on zeatin. Thus, Thidiazuron appears to stimulate enzyme activity converting the ribonucleotide to ribonucleoside. Although the cytokininactive phenylureas and adenine derivatives differ in their effects on cytokinin autonomy as well as nucleotide formation, the two types of effects do not seem to be related. 相似文献
17.
An enzyme mediating the conversion of zeatin to dihydrozeatin in phaseolus embryos 总被引:3,自引:2,他引:1 下载免费PDF全文
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 (Mr 55,000 ± 5,000) eluted as the second peak from the anion exchange column, while the low molecular weight isozyme (Mr 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. 相似文献
18.
Marta Kowalska Petr Galuszka Jitka Frébortová Marek Šebela Tibor Béres Tomáš Hluska Mária Šmehilová Kristin D. Bilyeu Ivo Frébort 《Phytochemistry》2010,71(17-18):1970-1978
The catabolism of cytokinins is a vital component of hormonal regulation, contributing to the control of active forms of cytokinins and their cellular distribution. The enzyme catalyzing the irreversible cleavage of N6-side chains from cytokinins is a flavoprotein classified as cytokinin dehydrogenase (CKX, EC 1.5.99.12). CKXs also show low cytokinin oxidase activity, but molecular oxygen is a comparatively poor electron acceptor. The CKX gene family of Arabidopsis thaliana comprises seven members. Four code for proteins secreted to the apoplast, the remainder are not secreted. Two are targeted to the vacuoles and one is restricted to the cytosol. This study presents the purification and characterization of each of these non-secreted CKX enzymes and substrate specificities are discussed with respect to their compartmentation. Vacuolar enzymes AtCKX1 and AtCKX3 were produced in Pichia pastoris and cytosolic enzyme AtCKX7 was expressed in Escherichia coli. The recombinant proteins were purified by column chromatography. All enzymes preferred synthetic electron acceptors over oxygen, namely potassium ferricyanide and 2,3-dimetoxy-5-methyl-1,4-benzoquinone (Q0). In slightly acidic conditions (pH 5.0), N6-(2-isopentenyl)adenine 9-glucoside (iP9G) was the best substrate for AtCKX1 and AtCKX7, whereas AtCKX3 preferentially degraded N6-(2-isopentenyl)adenine 9-riboside-5′-monophosphate (iPMP). Moreover, vacuolar AtCKX enzymes in certain conditions degraded N6-(2-isopentenyl)adenine di- and triphosphates two to five times more effectively than its monophosphate. 相似文献
19.
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. 相似文献
20.
Gametophore-over-producing mutants of the moss, Physcomitrella patens, when grown in liquid culture export high levels of cytokinin into their culture medium. The cytokinin produced by these mutants is postulated to account for their peculiar phenotype, that of mosses treated with exogenous cytokinin. N6-(Δ2-isopentenyl)adenine, the major cytokinin, has been identified previously in two of these mutants (Wang, Cove, Beutelmann, Hartmann 1980 Phytochemistry 19: 1103-1105) and now in additional representatives. A second cytokinin, zeatin, has been identified by its chromatographic behavior and mass spectrum including chemical ionization mass spectrometry of its permethyl derivative. 相似文献