A Cytokinin-binding Protein from Wheat Germ

Three auxin analogs, 4-, 5-, and 6-azido-3-indoleacetic acid (4N3-IAA, 5-N3-IAA, and 6-NA-IAA) have been synthesized for use as fluorescent photoaffinity labeling agents. The pKa values of these compounds (4-N3-IAA, 4.67; 5-Ns-IAA, 4.65; 6-N3-IAA, 4.66; all ± 0.04) are experimentally indisfinguishable from the pK. of 3-indoleacetic acid (IAA, 4.69 ± 0.04). The auxin activity of these IAA derivatives has been determined in several systems. In soybean, pea, and corn straight growth assays, all three analogs induce growth comparable to that caused by IAA. In the tobacco pith assay, anl three analogs elicit a maximum increase in fresh weight at least 40 to 50% of that caused by IAA. Optimal growth is attained in the tobacco pith assay at slightly higher concentrations of 4Ns-IAA and 6-N3-IAA (30 micromolar) than required for IAA (10 micromolar); however, maximal growth is achieved at a slightly lower concentration of 5-N3-IAA (3 micromolar). The NA-IAAs, like IAA, are transported basipetally through tobacco pith tissue.Auxins, the longest known class of plant hormones, modulate a wide variety of cell functions, the most significant being cell elongation (21,31,39,42,54,55). Despite extensive investigation, the mechanism of auxin action is still unknown, although it is now generally recognized that the response to auxins occurs in two phases (58,60,61). Within the past decade, reversible binding between auxins and several cell components has been demonstrated, establishing tentative locations of auxin receptors within the cell (17, 19, 23, 27, 43, 45, 50-52, 59, 61-66) (41).The decline in the rate of appearance of reports on auxin binding in the past 2 years compared to the rate during the 6 years immediately following the initial demonstration of auxin binding (23) and recent attempts to predict the properties of the auxin receptor from empirical structure-activity correlations (29,33,49) signal the need for a fresh approach to the problem of isolating an auxin receptor.To this end, we have synthesized three auxin analogs, 4-, 5-, and 6-azido-3-indoleacetic acid (4-N3-IAA, 5-N3-IAA, and 6-N3-IAA)7 for use as fluorescent photoaffinity labeling agents. The technique of photoaffinity labeling, which has been used to study animal hormones and to characterize the active sites of enzymes, has been thoroughly reviewed (6, 14-16, 30, 32

Section: Introductionmentioning

confidence: 99%

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confidence: 99%

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Azido Auxins: Synthesis and Biological Activity of Fluorescent Photoaffinity Labeling Agents

Melhado

Jones²,

Leonard³

et al. 1981

“…With few exceptions (16,21), attempts to locate and isolate plant hormone receptors have relied upon equilibrium binding techniques. Such techniques suffer inherent limitations, the most serious being the lability of ligand-receptor complexes.…”

Section: Discussionmentioning

confidence: 99%

Azido Auxins

Melhado¹,

Jones²,

Ho³

et al. 1984

The potential of three auxin analogs, 4-, 5-, and 6-azidoindole-3-acetic (4-N3IAA, 5-N3IAA, and 6-N31AA), as photoaffinity labeling agents for the detection and isolation of auxin receptors was assessed by irradiating these compounds at 365 nm on TLC plates, in solution, and in contact with soybean (Glycine max L. Meff. var. Wayne) hypocotyl. Photolysis on TLC plates produces immobile spots, indicating extremely polar or covalent binding of the photoproducts to the plates. On irradiation in buffer or in buffer containing sucrose, all three compounds decompose at rates that are first order in N3IAA to give fluorescent solutions. Photolysis through a Pyrex filter is slower than that through quartz, but the filter prevents tissue damage and allows a given dose of irradiation to photolyze all three N3IAAs to the same extent. The effects of photolysis of these compounds in vivo were evaluated with a straight growth assay using etiolated soybean hypocotyl segments. According to this assay, the photoproducts of the N3IAAs possess little auxin activity. Irradiation of soybean hypocotyl tissue after 1-hour exposure to 4-N3IAA in the dark causes the tissue to grow during 12 hours as much as tissue that is continuously exposed to 4-N3IAA in the dark for this period, suggesting that, on photolysis, this auxin analog binds irreversibly to an auxinsensitive site. Although the fluorescence intensity of the photolyzed N3IAAs is weak enough to require another method of detecting the bound analog under physiological conditions, the evidence for covalent binding of the N3IAAs on photolysis implies that these compounds will be satisfactory photoaffinity labeling agents.Earlier, we reported the synthesis of 4-, 5-, and 6-azido-3-indoleacetic acid and showed that, in the dark, these compounds are physiologically indistinguishable from IAA in every system tested (14). As a continuation ofthe assessment oftheir usefulness as fluorescent photoaffinity labeling agents, we now describe the photolysis of these compounds on TLC plates, in solution, and in vivo.

“…This protein has been extensively purified by (NHi>SO4 fractionation and repetitive gel filtration (19) and by combined ion exchange and affinity chromatographic procedures (7,14). While these preparations have similar cytokinin-binding properties, differences in apparent mol wt from gel filtration and differences in polypeptides present have been reported (7,9,14,19).…”

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confidence: 99%

Resolution and Properties of a Protein Kinase Catalyzing the Phosphorylation of a Wheat Germ Cytokinin-Binding Protein

Polya¹,

Davies²

1983

The nuaor cytoinin binig protde of wheat germ (CBP) was extensvely purired emloying chromatography on Cibacron F3GA-Sepharose CL and concanavain A-agarose as key purification steps. The mnor polypepides present In the purie CBP preparations have molcular weights of60,000 ± 4,000, 42,000 :t 3,000, and 37,000 ± 3,000, respectively. A protein kiae that catablzes the pbosphorylation of CBP (CBP kase) was extensively purified from wheat germ by affinity chromatograpby on casein-Sepharose 4B and CBP-Sepharose 4B. The pufication procedure resolves CBP kinase from an abundant casein kinase that does not pho_horylate CBP. CBP kinase catalyzes the phosphorylation of casein, pbosvitin, CBP, and the wheat germ cyclic AMP-binding protein cABPII.CBP khase posphorylates the major 60,000 dalton subunit of CBP as wel as 16,000 to 18,000 dalton polpptides present In CBP preparations.CBP fractions with differing activities as substrates for CBP khase were party resolved by gel ffltration and by chromatography on DEAE-Sepbacel.