Biomedical subjects

L L Melhado

Publications and source records attributed to L L Melhado.

5 recordsLinked to original sources

PubMed1984

Azido auxins : photolysis in solution and covalent binding to soybean.

The potential of three auxin analogs, 4-, 5-, and 6-azidoindole-3-acetic (4-N(3)IAA, 5-N(3)IAA, and 6-N(3)IAA), 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. Merr. 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 N(3)IAA 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 N(3)IAAs 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 N(3)IAAs possess little auxin activity. Irradiation of soybean hypocotyl tissue after 1-hour exposure to 4-N(3)IAA in the dark causes the tissue to grow during 12 hours as much as tissue that is continuously exposed to 4-N(3)IAA 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 N(3)IAAs is weak enough to require another method of detecting the bound analog under physiological conditions, the evidence for covalent binding of the N(3)IAAs on photolysis implies that these compounds will be satisfactory photoaffinity labeling agents.

Journal Article↗

PubMed1984

Azido auxins: quantitative binding data in maize.

The binding constants of three auxin analogs, 4-, 5-, and 6-azidoindole-3-acetic acid (4-, 5-, and 6-N(3)IAA), and of the photoproducts of 5-N(3)IAA to the naphthalene-1-acetic acid (NAA) binding sites of Zea mays L. WF9 x BR38 were determined to evaluate the potential of these analogs as photoaffinity labeling agents. We have found that 4- and 5-N(3)IAA bind to these sites with affinities similar to that of IAA, while 6-N(3)IAA and the photoproducts of 5-N(3)IAA bind less tightly. This binding is fully reversible in the dark. Binding of 5-N(3)IAA becomes covalent and irreversible upon UV irradiation, as evidenced by a 30% loss in NAA binding at sites pretreated with 5-N(3)IAA and UV irradiation, then washed extensively. IAA or NAA, included with this 5-N(3)IAA pretreatment, can protect the sites from blockage, whereas benzoic acid and tryptophan are unable to protect the site, indicating that 5-N(3)IAA specifically labels the auxin sites.

Journal Article↗

PubMed1984

Azido auxins : photoaffinity labeling of auxin-binding proteins in maize coleoptile with tritiated 5-azidoindole-3-acetic Acid.

Tritiated 5-azidoindole-3-acetic acid (5-N(3)-[7-(3)H]IAA), a photoaffinity labeling agent, was used to photolabel proteins of a crude microsomal preparation from maize (Zea mays L., Bear Hybrid, WF9 x BR38) coleoptile. Approximately 50% of the bound radioactivity was solubilized in 5 molar urea containing Triton X-100, and the extract was fractionated using a variety of techniques. High performance liquid chromatography demonstrated that, although many membrane proteins incorporated tritiated label, only a few showed reduced incorporation in the presence of excess indole-3-acetic acid. By contrast, no detectable reduction in incorporation was observed in the presence of excess naphthalene-1-acetic acid. Results from isoelectric focusing gel electrophoresis indicate that the proteins that showed reduced incorporation of photolyzed 5-N(3)-[7-(3)H]IAA in the presence of IAA fell into two main groups: one which focuses between pH 5.2 and 5.7 (pI 4.8-5.3) and another around pH 6.2 (pI 5.8). In sodium dodecylsulfate polyacrylamide gel electrophoresis, the proteins migrated as four bands with apparent molecular weights of 60, 49, 45, and 37 kilodaltons. The auxin-transport inhibitor, 2,3,5-triiodobenzoic acid, competes for the labeling by 5-N(3)-[7-(3)H]IAA, suggesting that some of these proteins may be involved in auxin transport.

Journal Article↗

PubMed1983

Rhodamine-based compounds as fluorogenic substrates for serine proteinases.

A new fluorogenic substrate for serine proteinases, bis(N-benzyloxycarbonyl-L-argininamido)Rhodamine [(Cbz-Arg-NH)2-Rhodamine], was synthesized, purified and chemically and enzymically characterized. This compound, which employs Rhodamine as a fluorophoric leaving group, is the first in a series of substrates designed to measure the amidase activity of proteinases. Cleavage of one of the amide bonds of (Cbz-Arg-NH)2-Rhodamine by a trypsin-like serine proteinase converts the non-fluorescent bisamide substrate into a highly fluorescent monoamide product. Significant differences in the electronic absorption and fluorescence emission spectra and quantum yields of bis-, mono- and un-substituted Rhodamine are reported. Macroscopic kinetic constants for the interaction of (Cbz-Arg-NH)2-Rhodamine with bovine trypsin, human and dog plasmin and human thrombin were determined. Compared with the corresponding 7-amino-4-methylcoumarin-based analogue, (Cbz-Arg-NH)2-Rhodamine exhibits an increase in sensitivity with these enzymes of 50--300-fold. The physical basis for this increase in sensitivity is discussed.

Chemical Phenomena↗

PubMed1981

Azido auxins: synthesis and biological activity of fluorescent photoaffinity labeling agents.

Three auxin analogs, 4-, 5-, and 6-azido-3-indoleacetic acid (4-N(3)-IAA, 5-N(3)-IAA, and 6-N(3)-IAA) have been synthesized for use as fluorescent photoaffinity labeling agents. The pK(a) values of these compounds (4-N(3)-IAA, 4.67; 5-N(3)-IAA, 4.65; 6-N(3)-IAA, 4.66; all +/- 0.04) are experimentally indistinguishable from the pK(a) 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, all 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 4-N(3)-IAA and 6-N(3)-IAA (30 micromolar) than required for IAA (10 micromolar); however, maximal growth is achieved at a slightly lower concentration of 5-N(3)-IAA (3 micromolar). The N(3)-IAAs, like IAA, are transported basipetally through tobacco pith tissue.

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