Triazine dyes, a new class of affinity labels for nucleotide-dependent enzymes

Clonis, Yannis D.; Lowe, Christopher R.

doi:10.1042/bj1910247

Cited by 93 publications

(65 citation statements)

References 15 publications

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“…6) is the dissociation constant k 2 / k l of the enzyme-dye complex [16,28]. The characteristics of the inhibition by the aminated, methoxy and hydroxy derivatives of Brown MX-5BR and their Ki values were different.…”

Section: Resultsmentioning

confidence: 99%

The Interaction of Tryptophanyl-tRNA Synthetase with the Triazine Dye Brown MX-5BR

1982

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Brown MX‐5 BR specifically and irreversibly inactivates tryptophanyl‐tRNA synthetase from Bacillus stearo‐thermophilus at pH 8.5. The enzyme is protected from inactivation by the substrates tryptophan and ATP and to lesser extents by ADP, AMP, the product inorganic pyrophosphate and other nucleotides such as GTP. The Kd of the pure reactive dye for the enzyme uah measured to be 6.7 × 10−5 M. The Km values of the two substrates tryptophan and MgATP were found to be 1 × 10−5 M and 5×10−5 M respectively. The aminated dye is a competitive inhibitor of tryptophanyl‐tRNA synthetase with respect to both tryptophan and MgATP with Ki values of 2×10−4 M against both substrates. The use of this dye as an active‐site‐directed affinity label is discussed.

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Section: Resultsmentioning

confidence: 99%

The Interaction of Tryptophanyl-tRNA Synthetase with the Triazine Dye Brown MX-5BR

1982

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“…This type of column is often used for enzymes containing nucleotide binding sites because of structural similarity with the dye structure [29]. The column (1 X 5 cm) of a cibacron-blueagarose was equilibrated and washed in buffer B.…”

Section: Step 1 Deae-sepharose Chromatographymentioning

confidence: 99%

Purification and characterization of the major tyrosine protein kinase from the human promyelocytic cell line, HL60

Ernould¹,

Ferry²,

Barret³

et al. 1993

European Journal of Biochemistry

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The major tyrosine protein kinase from HL60 (a human non-differentiated promyelocytic cell line) has been purified almost to homogeneity as judged by silver-stained SDS/PAGE. The procedure involved four chromatographic steps : DEAE-Sepharose, casein-agarose, cibacron-blue -agarose and hexyl-agarose. The purification resulted in more than 1000-fold enrichment in angiotensin I1 phosphorylation activity. A gel-sizing experiment, labeling with [35S]ATP [ys] and autophosphorylation of the enzyme in the presence of [Y-~~PIATP, all led to the identification of a single protein species with a molecular mass of about 40 kDa. Western blot experiments showed that this protein does not belong to the src family and is not related to the abl andfes oncogene products. Phosphorylation of angiotensin 11 and casein by this 40-kDa human promyelocytic kinase was stimulated by high ionic strength especially from class IA metal salts. The K, for ATP was 2 pM and the V,, 3.1 nmol min-' . mg-' using angiotensin fI as a substrate. The kinase requires the presence of either Mn2+ or Mg2+ for full activity and utilizes ATP or dATP but not GTP as phosphate donor. Based on numerous biochemical observations, it was possible to demonstrate that kinase is different from any other tyrosine protein kinases described in the literature. This 40-kDa protein was used as a molecular tool for testing some tyrosine protein kinase inhibitors described in the literature. It is one of the rare tyrosine protein kinases purified from human cancer cells to date.Protein phosphorylation plays a key role in the control of cellular communication [l]. Amongst the enzymes catalyzing the phosphorylation of other proteins (on specific amino acid residues), the tyrosine protein kinases (TPK) are known to be involved in the regulation of growth signal transmission and cellular transformation [2], according to their functional similarity to growth factor receptors and oncogene products [3]. A growing number of TPK(s) have been identified, mostly from molecular cloning of protein kinase genes rather than through enzyme purification [4]. TPK(s) can be classified in three categories [5][6][7]: (a) the transmembrane-receptor-associated TPK activity as found, for instance, in epidermal growth factor EGF, platelet growth factor PDGF and insulin receptors; (b) the TPK(s) linked to the inner face of the plasma membrane represented by the src family with src, yes, f g r , f m , lyn, hck, lck and blk oncogene products which are all myristoylated [8] and (c) the cytosolic TPK(s) with major representatives being abl and fes/fgr oncogene products.From a molecular pharmacological point of view, TPK(s) are new targets for inhibitors [9] To understand the cellular function of these TPKs one needs to characterize them on the basis of classical biochemistry studies (purification, molecular identification, kinetic data, effect of modulators, etc.). A major example was recently provided by Litwin et al. [24] and Cheng et al. [25], on a TPK specifically phosphorylating the p34cdc2 kinase ...

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“…phosphate dehydrogenase and yeast hexokinase at sites competitive with NAD', NADP+ and ATP respectively [16]. Furthermore, other triazine dyes have been shown to differentiate between the nucleotide binding sites of IMP dehydrogenase and adenylosuccinate synthetase [6] and to inactivate yeast hexokinase in an Mg'+-dependent fashion [17].…”

mentioning

confidence: 99%

“…The ability of a number of reactive monochloro or dichloro triazinyl dyes to mimic coenzyme binding has recently been exploited to devise effective irreversible affinity labels for nucleotide-dependent enzymes [16,17]. Thus several reactive dichlorotriazinyl dyes specifically and irreversibly inactivate pig heart lactate dehydrogenase, yeast glucose-6-Ahhrwutions.…”

mentioning

confidence: 99%

Affinity Labelling of Enzymes with Triazine Dyes

Small

Lowe

Atkinson

et al. 2005

European Journal of Biochemistry

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Horse liver alcohol dehydrogenase is irreversibly inactivated by Procion blue MX-R, a dichlorotriazinyl structural analogue of Cibacron blue F3G-A, with over 90% loss of activity within 30 min at pH 8.5 and 37°C at a reactive dye concentration of 1 mM and enzyme subunit concentration of 5 pM. Methoxylated Procion blue MX-R does not inactivate the enzyme. The inactivation of horse liver alcohol dehydrogenase by Procion blue MX-R is competitively inhibited by the pyridine nucleotides NAD' and NADH. Quantitatively inhibited horse liver alcohol dehydrogenase contains 1 mol dye/mol subunit of M , 40000. Chymotryptic digestion and resolution of the peptides by reverse-phase high-performance liquid chromatography yields a single blue peptide which on sequencing and analysis yields an amino acid sequence of:Procion blue MX-R

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Triazine dyes, a new class of affinity labels for nucleotide-dependent enzymes

Cited by 93 publications

References 15 publications

The Interaction of Tryptophanyl-tRNA Synthetase with the Triazine Dye Brown MX-5BR

The Interaction of Tryptophanyl-tRNA Synthetase with the Triazine Dye Brown MX-5BR

Purification and characterization of the major tyrosine protein kinase from the human promyelocytic cell line, HL60

Affinity Labelling of Enzymes with Triazine Dyes

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