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Several approaches were successfully performed to directly assign and characterize auxin binding of ABP44 in gel. The 44 kDa high affinity auxin binding protein ABP44 from pea was tested for its ability to bind 5-azido-[7-3H]-IAA in photoaffinity labeling experiments. Competition experiments with several auxin analogues confirm data published previously (Reinard and Jacobsen 1995). Critical reflections of the limitations of the method are also discussed. Immunostaining using the antibody D16 (Napier and Venis 1992), which is directed against the putative binding site of ABP1, revealed that ABP44's auxin binding site is at least partially related to the corresponding site of ABP1. Nevertheless, both proteins do not share any further immunological relationships. Our results with D16 recommend a careful reconsideration of data published by other authors. Furthermore, a 80 kDa, dimeric glutathione dependent formaldehyde dehydrogenase (FDH) from mung bean, described recently, was found to be different from ABP44. In contrast to the described FDH, ABP44 exhibited no FDH activity.

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In a search for membrane "docking proteins" interacting with Zea mays auxin-binding protein (ABP1) the binding of purified ABP1 to maize coleoptile plasma-membrane vesicles was investigated. Concentration-dependent, saturable binding of ABP1 to the membrane vesicles was observed in binding assays using 10(-8)-10(-6) M ABP1. Biotinylated ABP1 was displaced from the membrane binding sites by competition with unlabeled ABP1, demonstrating specific binding. The association step proved to be pH-dependent with maximum binding at pH 5.0 or lower. Auxins did not influence the ABP1 binding to plasma-membrane vesicles, but ABP1 associated with plasma-membrane vesicles was still able to specifically bind [3H]naphthalene-1-acetic acid. The rather stable interaction of ABP1 with plasma-membrane vesicles was only affected by strong alkaline buffers or detergents. The binding capacity was calculated to be in the range of 0.2 pmol ABP1 per g coleoptile fresh weight.

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Auxin-binding protein 1 (ABP1) is a unique hormone receptor because it resides primarily in the lumen of the endoplasmic reticulum (ER); however, two lines of evidence presented here suggest that ABP1 does not bind auxin within the endoplasmic reticulum, despite its predominant location there. First, ABP1 cannot be photolabeled in intact cells that have accumulated the auxin and photolabeling reagent 5-[7-3H]azidoindole-3-acetic acid, indicating either that auxin is excluded from the ER and is not available for photolabeling to ABP1 or that binding conditions within the ER lumen are insufficient for photolabeling. Second, at the pH of the ER lumen, auxin binding to ABP1 is not detectable. The pH estimate of the ER lumen is based on an indirect assay, which indicates that the pH is closer to pH 7 than to the binding optimum of pH 5.5. These results indicate that ABP1 does not bind auxin within the ER and point to a site of action that is post-ER. The effect of auxin on its trafficking from the ER was tested in an animal expression system. ABP1 expressed at high levels in COS7 cells is efficiently retained in the ER lumen and is not secreted even in the presence of 190 microM indole-3-acetic acid, an auxin concentration that is 40 times above the Kd for indole-3-acetic acid binding to ABP1.

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The auxin-binding protein isolated from maize coleoptiles and characterized in detail describes an auxin recognition protein at the outer surface of the plasmalemma which mediates the auxin effect on cell elongation in maize coleoptiles. Its homologue in tobacco mesophyll protoplasts mediates the auxin effect on secretion. The cDNA clones of the auxin-binding protein independently sequenced in three different laboratories contain one unique open reading frame describing the auxin-binding protein as a non-membrane-integrated glycoprotein containing the ER-sorting C-terminal tetrapeptide KDEL. There are hints but no hard facts that a membrane-located receptor for the ABP-auxin complex and a G-protein may be included in this signal-transducing pathway.

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The major auxin-binding protein from maize coleoptiles was purified to homogeneity. The protein has an apparent mol. wt of 22 kd and binds 1-naphthylacetic acid with a KD of 2.40 x 10(-7) M. Additional antigenically related proteins, present in very low amounts, could be demonstrated in maize coleoptiles using immunodetection. Extensive protein sequence analysis of the major auxin-binding protein allowed the construction of several synthetic oligonucleotide probes which were used to isolate a cDNA coding for this protein. The cDNA corresponds to a mRNA with a 3'-poly(A)+ sequence and a single, long open reading frame of 603 bases. The open reading frame, starting 34 residues from the 5' end of the cDNA, predicts a 21,990 Dalton protein of 201 amino acids. Comparison of this deduced amino acid sequence with the partial amino acid sequences of purified auxin-binding protein, revealed a perfect match, involving a total of 53 amino acid residues. The primary amino acid sequence includes a 38-amino-acid-long N-terminal hydrophobic leader sequence which could represent a signal for translocation of this protein to the endoplasmic reticulum. An additional signal is located at the C-terminal end, consisting of the amino acids KDEL known to be responsible for preventing secretion of proteins from the lumen of the endoplasmic reticulum in eucaryotic cells. The primary sequence contains a N-glycosylation site (-asp133-thr-thr-). This site was found to be glycosylated by a high-mannose-type oligosaccharide.(ABSTRACT TRUNCATED AT 250 WORDS)

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An auxin-binding protein (ABP) cDNA clone was selected from a lambda gt11 cDNA library from corn coleoptiles with highly purified IgGanti ABP. The sequence of 794 bp contains an open reading frame (ORF) of 603 bp, coding for a 22 kd protein. There are indications of a signal peptide of 38 amino acids (von Heijne, G. 1983, Eur. J. Biochem., 133, 17-21). A N-glycosylation site can be deduced and a C-terminal KDEL amino acid sequence is detected. An EcoRI fragment containing the beginning portion of the cDNA with about three quarters of the ORF was used to select cDNA clones from an independently produced lambda gt11 cDNA library of corn coleoptiles. Northern blot analysis with in vitro transcribed biotinylated RNA showed a single band of not more than 850 bases. The full-length in vitro transcript directed the in vitro synthesis of a protein which is precipitated by IgGanti ABP. Rabbit antibodies raised against a fusion protein detect the ABP as a double band on Western blots. Only the smaller of the two ABP bands is labeled by two different KDEL-specific IgG preparations.

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Tobacco mesophyll protoplasts were previously shown to respond to naphthaleneacetic acid by modifying their transmembrane potential difference. In the present work, evacuolated protoplasts were used to show that this response resides only at the plasmalemma. This electrical response was investigated by using polyclonal antibodies directed against plasma membrane antigens presumably involved in the reception and transduction of the auxin signal. An IgG fraction from an antiserum directed against the membrane auxin-binding protein from maize coleoptile completely inhibited the naphthaleneacetic acid-induced response of tobacco protoplasts. The suppression of the auxin-induced variation in the transmembrane potential difference by an IgG preparation directed against the plasmalemma ATPase from yeast demonstrated the involvement of the ATPase in the electrical response. Variation induced by fusicoccin in the transmembrane potential difference of tobacco protoplasts was unaffected by the anti-auxin-binding protein IgG fraction but was completely suppressed by the anti-ATPase IgG preparation. These results demonstrate the presence of a membrane receptor for auxin at the plasmalemma, the binding of the hormone to this receptor leading to the activation of the proton-pumping ATPase. They also show that at least the primary steps of activation by naphthaleneacetic acid are distinct from those of the fusicoccin-induced response.

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The purification of a putative auxin receptor is one possibility to elucidate the first event in the mechanism of auxin action. By affinity chromatography of membrane proteins on 2-OH-3,5-diiodobenzoic acid-Sepharose and gel filtration on Ultrogel a fraction enriched in auxin-binding protein (ABP) was obtained and used for rabbit immunization. From the immunoglobulin G (IgG) fraction of the antisera IgGs against proteins not binding auxin (nonABP) could be obtained which were used to eliminate the nonABP from the eluates of the 2-OH-3,5-diiodobenzoic acid-Sepharose. The remainder fraction was further purified and concentrated on IgG-Sepharose which retained the ABP that could be eluted without loss of binding activity. A 600-fold purification with a yield of 42% was achieved. The ABP could be identified as the site I "receptor" described by Dohrmann et al. (Dohrmann, U., Hertel, R., and Kowalik, H. (1978) Planta (Berl.) 140, 97-106). It is shown that the competitors tested reduce [14C]1-naphthylacetic acid-(NAA) binding in the following order of effectiveness: NAA greater than 2-naphthylacetic acid greater than 1-phenylacetic acid greater than 2,3,5-triiodobenzoic acid greater than 3-indolylacetic acid greater than 2,4-dichlorophenoxyacetic acid. The ABP has a sharp binding optimum at pH 5.5, and the KD was calculated to be 5.7 X 10(-8) M to [14C]NAA. The binding activity of the ABP linearly decreased with increasing temperature but could partially be restored upon chilling in the presence of auxin. The ABP seems to be a 40-kDa dimer in its native form without disulfide bonds between its monomers.

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With the aid of affinity chromatography on auxin-binding protein-Sepharose (ABP-Sepharose) monospecific IgGanti-ABP from rabbit antisera were isolated as judged by immuno-double diffusion test and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. With this IgGanti-ABP the ABP is localized within the outer epidermal cells of coleoptiles using indirect immunofluorescence labeling. Auxin-induced growth of coleoptile segments can be inhibited by IgGanti-ABP, and the auxin response of split coleoptile sections is also strongly reduced by IgGanti-ABP. The ABP, therefore, is referred to as an auxin receptor. This auxin receptor is localized at the plasmalemma of the outer epidermal cells of the coleoptile.

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Roots of intact bean plants were supplied with [(14)C]adenine by pulse-chase experiments. The rate of incorporation of radioactivity into tRNA and oligonucleotides of roots as well as the content of radioactive labeled cytokinin nucleotides in these RNA fractions were determined. On the average, 1/70 of the radioactivity incorporated into tRNA was localized in N(6)(Δ(2)isopentenyl)adenosine. The half life of tRNA was estimated to be 65-70 h. Shortly after the pulse period, oligonucleotides contained zeatin riboside at a ratio of 1:800, on the basis of radioactivity. The half life of these oligonucleotides was determined to be about 8 h. The main free radioactive cytokinin of roots and leaves was zeatin. Comparing the rate of degradation of (14)C-labeled tRNA and the oligonucleotides of roots and the rate of appearance of radioactive cytokinins in roots and leaves, we found strong indications for their dependency. The results contradict the hypothesis of de novo synthesis of cytokinins in roots of intact bean plants.

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The extraction and purification of the tRNA isopentenyltransferase from maize root tips and kernels are reported. The relative amounts of this enzyme in different organs of maize have been determined. Root tips have the highest enzyme activities, followed by kernels and young leaves. Old leaves exhibit very low activity. The molecular mass of the monomeric enzyme was determined to be 57000 - 63000 dalton. pH and Mg2 optima are in full agreement with the data reported for the enzymes from yeast and Escherichia coli. Spermine enhances the isopentenylation of tRNA from kernels. The "Km" values of KMnO4-treated or untreated bulk tRNA from yeast and maize root tips and kernels were in the range of 10 to 34 micrometer while the Km values of of single species like tRNASer4 from rat liver and tRNATyrKMnO4 from E. coli were 1.1 and 6.6 micrometer, respectively. The tRNA isopentenyltransferase from maize root tips and kernels catalyzes the incorporation of 2-isopentenyl groups into (Ap)3-7A, endogenous bulk oligonucleotos from maize root tips and kernels, and into poly(A), RNA from MS-2 phages and, to a very low extent, into adenosine. The Km values of (Ap)3-7 A varied in the range of 250 to 750micrometer. Although oligonucleotides have less affinity to the enzyme, the formation of i6A within oligonucleotides may occur in vivo, due to their higher concentrations in cells.

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The influence of naphthalene-1-acetic acid and kinetin on protein synthesis in vivo was investigated by measuring the incorporation of radioactive amino acids into polypeptides of synthesizing polysomes. The second subculture of sterile pith tissue of Nicotiana tabacum L. cv. Wisconsin 38 grown on a medium containing only a minimum of growth substances was further starved in a small volume of medium lacking auxin and cytokinin for 12 h. An incubation period of 4 h with [(14)C]amino acids followed. The last 30 min of those incubations were carried out in the presence of actinomycin D and the last 20 min were performed under different conditions: a) without any growth substances, b) with naphthalene-l-acetic acid, c) with kinetin. By measuring the differences of the specific radioactivities of the polysomes the following results were revealed: 1) Kinetin increases the protein synthesis by an average of 35%-2) Auxin has no effect on protein synthesis.

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Two protein factors, isolated from S-100 of maize shoots, were found to be required not only for a "Mg(2+) shift" for poly U-dependent polymerization of phenylalanine, but also for polyphenylalanine synthesis. They seem to be crude preparations of EF-1 and EF-2. The poly U-directed amino acid incorporation by washed ribosomes was completely dependent upon these factors.

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The influence of auxin and cytokinin on RNA synthesis was investigated by measuring (32)PO4 (3-)-incorporation into polysomes and polysomal RNAs of tissue grown from pith of Nicotiana tabacum L. The test tissue grown on a medium containing a minimum of auxin and cytokinin was further starved for a period of 12 h prior to the experiments. The tests were carried out under the following different conditions: 1. without any growth substances, 2. with auxin only, 3. with kinetin only, and 4. with auxin and kinetin. The tissues were incubated in small volumes of the test medium for 1-8 h on a shaker. The last hour of incubation was carried out in the presence of (32)PO4 (3-).Differences in (32)PO4 (3-) uptake into the polysomes were observed as early as after 3 h of incubation. Kinetin alone shows no effect at all, auxin causes increased synthesis of polysomal RNAs, and kinetin administered together with auxin augments the increase of (32)PO4 (3-) uptake into polysomes and polysomal RNAs observed with auxin alone. These results and those of Seth and Wareing [J. exp. Bot. 18, 65-77 (1967)], Bhattacharyya and Roy [Biochem. biophys. Res. Commun. 35, 606-610 (1969)], and Venis [Proc. nat. Acad. Sci. (Wash.) 68, 1824-1827 (1971)] lead to the following working hypothesis: while auxins exert their influence on the RNA transcription, cytokinins may possibly influence the translation process.

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An amino acid incorporating system has been prepared from maize seedlings, and it has been characterized with the aid of poly-U and a "mRNA enriched fraction" from the same plant material.The rate of protein synthesis decreases proportionally with the incubation period. It seems to be related to the degradation of polysomes. The optimal Mg(2+) concentration is 20 mM for the poly-U dependent protein synthesis and 10 mM for the synthesis with endogenous polysomes. The poly-U directed polyphenylalanine synthesis is increased 12-fold by addition of exogenous sRNA. Under optimal conditions poly-U causes a 40-fold increase of the phenylalanine incorporation.A "mRNA enriched fraction" was prepared from maize seedlings using proteinase K for deproteination of polysomes. The resulting RNA was further fractionated by successive precipitation with LiCl, NaCl and ethanol and characterized by polyacrylamide gel electrophoresis. The addition of 57 µg of the mRNA-enriched sample increases the incorporation of amino acid into polypeptides by a factor of approximately 2 at a Mg(2+) concentration of 5 mM, and by a factor of 1.5 at 15 mM Mg(2+). The addition of 72 µg rRNA does not stimulate the incorporation at low Mg(2+) concentration, while at 15 mM Mg(2+) a 1.3-fold increase is observed.

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The uptake into wheat coleoptile tissue of C-14 radioactivity from naphthalene-1-acetyl-aspartic acid-C-14 (NAAS) compared with that from naphthalene-1-acetic acid-C-14 (NES) is as low as 1:10. The data give strong evidence for an active uptake of NES and even NAAS. A comparison of the NES uptake with the growth response within the first 3 hours indicates that the optimal concentration of auxin for maximal growth rate is about 2 mµMol/hour/g of fresh weight. NAAS acts as an auxin only after hydrolysis to NES. Considering the fact that the tissues show no growth response to NAAS during the first hours of incubation even though the C-14 uptake from this substance is as much as one tenth of that from NES, the hydrolysis of NAAS to NES must take place inside the cells. Chromatographic studies of wheat coleoptile segments incubated in NAAS-C-14 confirm this conclusion.

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Wheat coleoptile segments were pulse labeled with C-14-proline (pro) for different 2-hour periods within a total incubation of 8 hours. The effect of auxin naphthalene-1-acetic acid (NAA) and hydroxyproline (hypro) on the pro incorporation into cell wall fraction and soluble proteins was measured. 1. NAA causes a reduced incorporation of C-14-pro into soluble proteins and into the cell wall fraction within the first 2-hour period. Pulse labeling at later periods results in a marked increase of C-14-pro incorporation into the cell wall fraction due to NAA without change in the incorporation rate into soluble proteins. 2. There is no influence of NAA or hypro on the hydroxylation of pro to hypro. 3. From the second to the sixth hour of incubation the increase of pro incorporation into the cell wall fraction effected by auxin is closely correlated with the stimulation of cell expansion growth. 4. Hypro in growth inhibiting concentration (10(-2)M) does not show a clear influence upon pro hydroxylation, but it does have a remarkable effect in reducing auxin stimulated pro incorporation into the cell wall fraction From the data given it can be assumed that auxin may exert a direct function in regulating cell wall protein synthesis.