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D Klämbt

Publications and source records attributed to D Klämbt.

14 recordsLinked to original sources
PubMed1998

Assignment of the auxin binding abilities of ABP44 in gel.

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.

Gels↗
PubMed1997

Interaction of auxin-binding protein 1 with maize coleoptile plasma membranes in vitro.

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.

Cell Membrane↗
PubMed1995

Auxin-binding protein 1 does not bind auxin within the endoplasmic reticulum despite this being the predominant subcellular location for this hormone receptor.

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.

Animals↗
PubMed1990

A view about the function of auxin-binding proteins at plasma membranes.

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.

Cell Membrane↗
PubMed1989

Molecular cloning and structural analysis of a gene from Zea mays (L.) coding for a putative receptor for the plant hormone auxin.

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)

Amino Acid Sequence↗
PubMed1989

cDNA clones of the auxin-binding protein from corn coleoptiles (Zea mays L.): isolation and characterization by immunological methods.

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.

Base Sequence↗
PubMed1989

Functional evidence for an auxin receptor at the plasmalemma of tobacco mesophyll protoplasts.

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.

Journal Article↗
PubMed1985

Auxin-binding protein from coleoptile membranes of corn (Zea mays L.). I. Purification by immunological methods and characterization.

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.

Animals↗
PubMed1985

Auxin-binding protein from coleoptile membranes of corn (Zea mays L.). II. Localization of a putative auxin receptor.

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.

Chromatography, Affinity↗
PubMed1978

tRNA isopentenyltransferase from Zea mays L. Characterization of the isopentenylation reaction of tRNA, oligo (A) and other nucleic acids.

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.

Adenosine↗