Anthracenone ABA analogue as a potential photoaffinity reagent for ABA-binding proteins

An anthracenone analogue of abscisic acid (ABA) was synthesized as a potential photoaffinity reagent and tested for biological activity. Reaction between 10,10'-dimethoxy-9-anthrone with two equivalents of the lithiated dianion of cis-3-methylpent-2-en-4-yn-1-ol afforded an acetylenic alcohol key intermediate. Subsequent reduction of the triple bond, functional group manipulation of the side chain alcohol and deprotection of the dimethoxy protected anthrone provided anthracenone ABA analogue 7 as a potential photoaffinity reagent for ABA-binding proteins. The effect of natural ABA and the potential photoaffinity anthracenone ABA 7 on corn cell growth was determined at various concentrations. The results show that anthracenone ABA 7 is perceived as ABA-like, although producing less inhibition than ABA itself. For example, 7 at 33 microM produces approximately the same inhibition as ABA at 10 microM.     

Azido derivative of tricarboxylic acid for photoaffinity labeling

A new photoaffinity probe, 5-(1-hydroxy-4-azidophenylazo)-1,2,3-benzenetricarboxylic acid, was synthesized and characterized. This reagent can be potentially used in photoaffinity labeling of the mitochondrial tricarboxylate carrier, as well as of enzymes interacting with tricarboxylic acids. Inhibition and labeling of the mitochondrial tricarboxylate carrier is presented.     

Synthesis of 5-nitro-2-[N-3-(4-azidophenyl)-propylamino]-benzoic acid: photoaffinity labeling of human red blood cell ghosts with a 5-nitro-2-(3-phenylpropylamino)-benzoic acid analog

A photoaffinity analog of the potent epithelial chloride channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoic acid has been synthesized and characterized. In the dark, this reagent, 5-nitro-2-[N-3-(4-azidophenyl)-propylamino]-benzoic acid, and the parent compound reversibly inhibited chloride efflux in human red blood cell ghosts. Irradiation of ghost membranes with 350 microM arylazide analog reduced the rate of chloride efflux to 33% of the control value. The photoinactivation process was not reversed by exhaustive washing of ghost membranes. Covalent incorporation of the photoaffinity reagent was supported by difference ultraviolet spectroscopy, which indicated the attachment of the substituted 2-amino-5-nitrobenzoic acid chromophore to ghost membranes. The novel photolabeling agent described here should be a useful structural probe for chloride channels in erythrocyte membranes and epithelial cells.     

An azidoaryl thioglycoside of sialic acid. A potential photoaffinity probe of sialidases and sialic acid-binding proteins

An azidoaryl thioglycoside of sialic acid was prepared, as a potential photoaffinity probe reagent for the analysis of sialidases and sialic acid-binding proteins, by treatment of the glycosyl chloride of N-acetylneuraminic acid methyl ester with potassium thioacetate to give, in 70% yield, methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-2-S-acetyl-2,3,5-trideoxy-2-thio-alph a-D- glycero-D-galacto-2-nonulopyranosonate. Selective hydrolysis of the thioacetate ester, followed by condensation with 4-fluoro-3-nitrophenyl azide, O-deacetylation, and hydrolysis gave (4-azido-2-nitrophenyl)- 5-acetamido-2,3,5-trideoxy-2-thio-alpha-D-glycero-D-galacto-2- nonulopyranosidonic acid.     

Photoaffinity labeling and mass spectrometry identify ribosomal protein S3 as a potential target for hybrid polar cytodifferentiation agents.

The ability of a novel class of hybrid polar compounds (HPCs) to induce differentiation and consequent cessation of proliferation of transformed cells has led to their development as potential chemotherapeutic agents in the treatment of cancer. Suberoylanilide hydroxamic acid (SAHA) is a prototype of a family of hydroxamic acid based compounds (SAHA-like HPCs) that can, at micromolar concentrations, induce a variety of transformed cell lines to differentiate. The mechanism of action of the HPCs is not entirely understood. Searching for a cellular target of the SAHA-like HPCs, we synthesized a photoaffinity labeling reagent structurally based on SAHA, and probed for SAHA-binding proteins in murine erythroleukemia (MEL) cells. Photoaffinity labeling in cell free extracts identified a 32-kDa protein (p32) that was specifically labeled by the photoaffinity reagent. Cell fractionation assays localized p32 to the P100 fraction. p32 was partially purified and identified by mass spectrometry as the 40 S ribosomal protein S3. Expression of epitope-tagged S3 in bacterial lysates followed by photoaffinity labeling confirmed its specific labeling. Identification of a cytodifferentiation agent target may shed light on the mechanism by which the SAHA-like HPCs exert their antitumor effects.     

Photoaffinity labeling of specific alpha-thrombin binding sites on Chinese hamster lung cells

Binding sites for alpha-thrombin on cultured Chinese hamster lung cells were identified using photoactivatable cross-linking conjugates of diisopropylphosphorofluoridate-inactivated alpha-thrombin. A series of photoaffinity reagents was synthesized that permitted systematic variation of the extent of thrombin modification and of steric factors affecting the ability of the photoaffinity reagent to contact thrombin receptors. The reagents were synthesized with a tritium label to accurately determine the number of photoaffinity molecules linked to each thrombin. Also, they were synthesized with different length spacer arms between the photoreactive cross-linking group (a nitroarylazide) and the end which linked to alpha-thrombin (a succinimide ester). By calculating the percentage of the thrombin surface that would be accessed by modifying it with a fixed molar excess of each reagent, it was possible to select the photoaffinity reagents that would be most effective for cross-linking 125I-labeled diisopropylphosphorofluoridate-inactivated alpha-thrombin to its cellular binding sites. The validity of this selection procedure was confirmed in experiments in which an Mr = 150,000 cellular component was labeled. This component had the properties of a specific binding site for thrombin since labeling was readily competed for by nonlabeled alpha-thrombin. The cross-linking achieved was due to the photoactivatable reagent since no detectable cross-linked complex was formed in the absence of photoactivation or with 125I-labeled diisopropylphosphorofluoridate-inactivated alpha-thrombin that was not conjugated with the photoaffinity reagent.     

Use of a solid-phase photoaffinity reagent to label a steroid binding site: application to the delta 5-3-ketosteroid isomerase of Pseudomonas testosteroni

In order to extend our analysis of the reactions that occur during the active site directed photoinactivation of delta 5-3-ketosteroid isomerase sensitized by unsaturated steroid ketone photoaffinity reagents, the site of covalent attachment has been identified. A solid-phase photoaffinity reagent, delta 6-testosterone-agarose, has been employed for this purpose; this type of reagent, in contrast to solution-phase reagents, facilitated the recovery of a peptide fragment of the isomerase bearing the residue at which covalent attachment had occurred. Amino acid analysis and sequence determination of the peptide provided evidence that the site of attachment was aspartate-38. This result, in combination with the low-resolution crystallographic structure of the enzyme [Westbrook, E. M., Piro, O. E., & Sigler, P. B. (1984) J. Biol. Chem. 259, 9096-9103], suggests that aspartate-38 is located in the vicinity of the bottom of the steroid-binding pit. The potential usefulness of solid-phase photoaffinity reagents in the identification of sites of covalent attachment on target proteins such as hormone receptors is discussed.     

A stable diazo photoaffinity label with high absorptivity and effective photoactivation beyond 300 nm.

The sulfosuccinimidyl active ester of 3-(3-carbethoxy-4-diazo-5-oxo-2-pyrrolin-1-yl)propanoic acid (DIAZOPY-SE) has been synthesized for use as a photoaffinity labeling reagent. This compound was obtained from commercial chemicals by a four-step synthesis requiring no complex procedures or special apparatus. The active ester efficiently derivatizes protein amino groups with the chromophore 3-carbethoxy-4-diazo-5-oxo-2-pyrroline (DIAZOPY, epsilon 8800 M-1 cm-1 at lambda max 330 nm), which on irradiation yielded products expected from formation of a reactive carbene intermediate. Brief irradiation of DIAZOPY in 2-propanol using wavelengths greater than 300 nm for photolysis yielded mainly an isopropyl ether resulting from insertion of the carbene into the O-H bond of the alcohol. Formed concurrently and to a somewhat lesser extent was an isopropyl ester, resulting from a ring-contracting Wolff rearrangement of the carbene and subsequent reaction with isopropanol. Analogous products were produced by photolysis in 2-propanol of DIAZOPY-PA (for DIAZOPY propanoic acid), the carboxylic acid precursor of DIAZOPY-SE. Facile protein derivatization by DIAZOPY-SE was demonstrated using actin and sheep IgG. Actin labeled with DIAZOPY-SE and irradiated while in the F-actin (reversibly polymerized) form was crosslinked to yield a covalently-linked dimer, illustrating the potential of the reagent in photoaffinity applications. Advantages of DIAZOPY-SE as a photoaffinity labeling reagent include ease of synthesis, chemical and photostability, efficient photolysis at wavelengths greater than 300 nm, and a capacity for crosslinking by carbene insertion processes.     

New developments in abscisic acid perception and metabolism

Abscisic acid is a powerful signaling molecule that accumulates in response to abiotic stress. However, no potential receptors that could perceive this increase in abscisic acid had been identified until recent reports of three abscisic acid binding proteins: the nuclear protein Flowering Time Control Locus A, the chloroplast protein Magnesium Protoporphyrin-IX Chelatase H subunit, and the membrane-associated protein G Protein Coupled Receptor 2. Abscisic acid metabolism also has a new and prominent component with the identification of a beta-glucosidase capable of releasing biologically active abscisic acid from inactive abscisic acid-glucose ester in a stress-inducible manner. These observations refocus our attention on the metabolism underlying abscisic acid accumulation, sites of abscisic acid perception, and delivery of abscisic acid to those sites.     

Photoaffinity labeling of human retinoid X receptor beta (RXRbeta) with 9-cis-retinoic acid: identification of phytanic acid, docosahexaenoic acid, and lithocholic acid as ligands for RXRbeta

We utilized [20-methyl-(3)H]-9-cis-retinoic acid ([(3)H]9-cis-RA) as a direct photoaffinity probe for the characterization of human recombinant retinoid X receptor beta protein (RXRbeta). The photoaffinity labeling was light- and concentration-dependent, saturable, and protected by unlabeled 9-cis-RA in a concentration-dependent manner, indicating that binding occurred in the RXR retinoid binding site. all-trans-Retinoic acid (atRA) did not affect labeling with the 9-cis derivative, confirming that atRA does not compete for the 9-cis-RA binding site. Several retinoid, fatty acid, and bile acid ligands were evaluated for their ability to recognize the 9-cis-RA binding site. Retinol, atRA glucuronide, 13-cis-RA, dolichol, 5,6-epoxy-RA, and vitamin D(3) did not compete for the 9-cis-RA binding site. However, the saturated diterpenoid phytanic acid (PA) and docosahexaenoic acid, which have been recently shown to activate the nuclear receptor, RXR, competed with 9-cis-RA labeling, showing high affinity for the 9-cis-RA binding site. Oleic acid, arachidonic acid, and butyric acid did not interact. However, the bile acid lithocholic acid competed efficiently with 9-cis-RA for the binding site. These data validated the photoaffinity assay as an excellent system for the identification and evaluation of ligands for RXR.     

An enzyme-immunoassay for cis-(+)-abscisic acid

A solid-phase enzyme-immunoassay for abscisic acid and abscisic acid conjugates in fmol amounts (detection limit ca 50–60 fmol) has been developed. Only little procedural effort is required for the assay which can be completed within 6 h. With this method, abscisic acid was detected in a range of heterotrophically growing plant suspension cultures. In addition, abscisic acid-glucosyl ester was identified in a Lonicera prolifera Rehd. tissue culture. In this tissue, the level of free abscisic acid reaches ca 2 × 10⁻⁶ M in the late log-phase of growth.     

Photoaffinity labeling of GDP-fucose:nLcOse4Cer alpha 1----3-fucosyltransferase from human small cell lung carcinoma NCI-H69 cells with the GDP-fucose analog GDP-hexanolaminyl-4-azidosalicylic acid

An iodinatable photoactive analog of GDP-fucose, GDP-hexanolaminyl-4-azidosalicylic acid, has been prepared and applied to studies of the previously described alpha 1----3-fucosyltransferase from NCI-H69 cells (Holmes, E. H., Ostrander, G. K., and Hakomori, S. (1985) J. Biol. Chem. 260, 7619-7627). The NCI-H69 cell alpha 1----3-fucosyltransferase was obtained from a 0.2% Triton X-100-solubilized enzyme fraction after affinity purification on a GDP-hexanolamine-Sepharose column and gel filtration through a fast protein liquid chromatography Superose 12 column. Increasing concentrations of the photoaffinity reagent were found to result in loss of up to 35% of the original enzyme activity at under 100 microM final concentrations. The inactivation was photolysis dependent and could be prevented by the addition of GDP-fucose prior to photolysis. The photoprobe behaved as a competitive inhibitor with respect to GDP-fucose with a Ki of 23 microM, identical to that of GDP. Photoincorporation of 125I-labeled GDP-hexanolaminyl-4-azidosalicylic acid into the enzyme fraction labeled a slow migrating protein band in a native polyacrylamide gel which corresponded to enzyme activity. Inclusion of GDP-fucose prevented photolabeling of this band. Sodium dodecyl sulfate gel electrophoresis of the photolabeled, GDP-fucose-protected band yielded a 125I-labeled protein band that migrated at Mr 45,000, most probably corresponding to an alpha 1----3-fucosyltransferase protein subunit. These studies suggest photoaffinity labeling using nucleotide affinity ligands linked to photoactivatable, heterobifunctional cross-linking reagents may be generally applicable to photoaffinity labeling glycosyltransferase enzyme proteins.     

The role of abscisic acid in plant-pathogen interactions

The effect of the abiotic stress hormone abscisic acid on plant disease resistance is a neglected field of research. With few exceptions, abscisic acid has been considered a negative regulator of disease resistance. This negative effect appears to be due to the interference of abscisic acid with biotic stress signaling that is regulated by salicylic acid, jasmonic acid and ethylene, and to an additional effect of ABA on shared components of stress signaling. However, recent research shows that abscisic acid can also be implicated in increasing the resistance of plants towards pathogens via its positive effect on callose deposition.     

Synthesis of the photoaffinity probe 3-(p-azidobenzyl)-4-hydroxycoumarin and identification of the dicoumarol binding site in rat liver NAD(P)H:quinone reductase (EC 1.6.99.2).

A photoaffinity analog of 4-hydroxycoumarin containing an azidobenzyl group at the 3-position and, if desired, carbon-14 or tritium radionuclides has been synthesized and characterized. This compound, 3-(p-azidobenzyl)-4-hydroxycoumarin, serves as an effective competitive inhibitor of the dicoumarol-sensitive NAD(P)H:quinone reductase (EC 1.6.99.2; DT-diaphorase) from rat liver, having an apparent inhibition constant of 6.6 x 10(-8) M, a value comparable to that observed for dicoumarol (1.7 x 10(-9) M), significantly lower than for Warfarin (3.5 x 10(-5) M) and well within the range required of an effective photoaffinity reagent. Irradiation of the reductase with ultraviolet light in the presence of the photoprobe resulted in the covalent labeling of up to 10% of the protein. Greater than 99% of the covalent incorporation is precluded by the addition of 15 microM dicoumarol, consistent with the specific labeling of the 4-hydroxycoumarin binding site of this enzyme by this photoaffinity reagent. Further evidence of a high degree of specificity is provided by the isolation and sequence analysis of the peptides covalently modified by this reagent. A single region within the protein was found to be labeled, with threonine 127 and tyrosine 128 being the only amino acid residues that were observed to be modified. These results, for the first time, define a portion of the 4-hydroxycoumarin binding site within a protein that has a well established sensitivity to this type of anticoagulant and, because dicoumarol serves as a competitive inhibitor for pyridine nucleotides in this enzyme, may also define a portion of this unusual pyridine nucleotide binding site. In addition, these results suggest that this reagent may be effective as a highly specific photoaffinity probe in the identification of other proteins that are similarly inhibited by 4-hydroxycoumarin derivatives, such as the microsomal enzymes associated with the vitamin K-dependent carboxylation system.     

Further evidence of a role for abscisic acid in conversion of somatic embryos ofDaucus carota

Summary Somatic embryogenesis has been shown to be an imperfect recapitulation of stages involved to form embryos from vegetative tissues. Although abscisic acid has been implicated in normalizing development, studies that specifically investigate conversion (vegetative leaf initiation) in somatic embryos are lacking. This report documents a follow-up of a study that implicated abscisic acid as a vital factor in allowing embryos ofDaucus carota to progress to the plantlet stage. Abscisic acid was determined to enhance conversion at doses ranging from 1 to 50 µM. Younger embryo stages were more responsive to abscisic acid application with regards to plantlet recovery. Pulses of abscisic acid were shown to elicit more rapid response with younger embryo stages, indicating more plastic development. Fluridone, an abscisic acid synthesis inhibitor, was shown to dramatically reduce conversion, even at low doses (<5µM). When abscisic acid was applied concurrently with fluridone, partial restoration of conversion was observed. Histologically, fluridone was seen to cause pronounced vacuolation in the shoot apical notch which resulted in the loss of meristematic cells, negating conversion capacity. Quantitation of total cytoplasmic area showed that abscisic acid reduced vacuolar intrusion into the apical notch, while fluridone caused a significant increase in vacuolation of cells in this region. This report documents further evidence of a role for abscisic acid in plantlet establishment from somatic embryos ofDaucus carota.     

The directional control of sucrose and asparagine transport in lupin by abscisic acid

Injections of exogenous abscisic acid into the primary flowerhead of Lupinus luteus cv. Weiko III reduced the movement of ¹⁴C-sucrose into the flowerhead from the uppermost leaves. Sucrose transported from below the lateral branches subtending the flowerhead, was diverted into the lateral branches by injection of the exogenous abscisic acid into the flowerhead. ¹⁴C-sucrose was also diverted from a lateral branch injected with exogenous abscisic acid to all other parts of the plant, particularly the main stem and leaves, and the roots. Transport of ¹⁴C-asparagine administered at the cotyledonary node was directed from the flowerhead into the subtending lateral branches by injection of abscisic acid into the flowerhead. Transport of both ¹⁴C-sucrose and ¹⁴C-asparagine into the flowerhead was reduced at least three fold at physiological levels of abscisic acid. No significant correlation was found between the amount of ¹⁴C-asparagine entering a sink and the dry weight of the tissues of that sink. It is concluded that distribution of ¹⁴C-sucrose and ¹⁴C-asparagine between the flowerheads and lateral branches of L. luteus is actively and dynamically controlled and that abscisic acid levels play a significant part in that control. It is suggested that the relative levels of endogenous abscisic acid in plant organs could serve as an important factor in the directional control of assimilate transport in plants.     

Carotenogenic and abscisic acid biosynthesizing activity in a cell-free system

Abscisic acid is considered an apocarotenoid formed by cleavage of a C-40 precursor and subsequent oxidation of xanthoxin and abscisic aldehyde. Confirmation of this reaction sequence is still awaited, and might best be achieved using a cell-free system capable of both carotenoid and abscisic acid biosynthesis. An abscisic acid biosynthesizing cell-free system, prepared from flavedo of mature orange fruits, was used to demonstrate conversion of farnesyl pyrophosphate, geranylgeranyl pyrophosphate and all-trans-β-carotene into a range of β,β-xanthophylls, xanthoxin, xanthoxin acid, 1′,4′-trans-abscisic acid diol and abscisic acid. Identification of product carotenoids was achieved by high-performance liquid chromatography and on-line spectral analysis of individual components together with co-chromatography. Putative C-15 intermediates and product abscisic acid were identified by combined capillary gas chroma-tography-mass spectrometry. Kinetic studies revealed that β-carotene, formed from either famesyl pyrophosphate or geranylgeranyl pyrophosphate, reached a maximum within 30 min of initiation of the reaction. Thereafter, β-carotene levels declined exponentially. Catabolism of substrate β-carotene into xanthophylls, putative abscisic acid precursors and product abscisic acid was restricted to the all-trans-isomer. However, when a combination of all-trans- and 9-cis-β-carotene in the ratio 1:1 was used as substrate, formation of abscisic acid and related metabolites was enhanced. Biosyn-thetically prepared [¹⁴C]-all-trans-violaxanthin, [¹⁴C]-all-trans-neoxanthin and [¹⁴C]-9′-cis-neoxanthin were used as substrates to confirm the metabolic interrelationship between carotenoids and abscisic acid. The results are consistent with 9′-cis-neoxan-thin being the immediate carotenoid precursor to ABA, which is oxidatively cleaved to produce xanthoxin. Formation of abscisic aldehyde was not observed. Rather, xanthoxin appeared to be converted to abscisic acid via xanthoxin acid and 1′,4′-trans-abscisic acid diol. An alternative pathway for abscisic acid biosynthesis is therefore proposed.     

Asparagusic acid, a new plant growth inhibitor in etiolated young asparagus shoots

Yellow prisms of asparagusic acid, with a molecular formulaof C₄H₆O₂S₂ were isolated from etiolated asparagus tissues (Asparagusofficinalis L.). This acid inhibits growth in lettuce and otherseedlings when applied in concentrations of 6.67x10^–7Mto 6.67xl0^–7M. The extent of activity was very similarto that of abscisic acid. ¹ A well known shift reagent in the NMR spectrum (1). (Received April 12, 1972; )     

Photoaffinity labelling of the 2-oxoglutarate binding site of prolyl 4-hydroxylase with 5-azidopyridine-2-carboxylic acid

The synthesis of the photoaffinity label 5-azidopyridine-2-carboxylic acid is described. The 2-oxoglutarate analogue photoaffinity label is a competitive inhibitor with respect to 2-oxoglutarate with a Ki value of 9 X 10(-3) M. Upon ultraviolet irradiation, 5-azidopyridine-2-carboxylic acid inactivated prolyl 4-hydroxylase irreversibly by up to 50%. The extent of inactivation depended on the 5-azidopyridine-2-carboxylic acid concentration and the irradiation time. Inactivation was prevented in the presence of an excess of 2-oxoglutarate. It is concluded that the 5-azidopyridine-2-carboxylic acid became covalently bound to the alpha subunit of prolyl 4-hydroxylase, as the alpha subunit of the photoaffinity labelled enzyme had a decreased electrophoretic mobility in polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate.