Bimane fluorescent labels: labeling of normal human red cells under physiological conditions.

Kosower, Nechama S.; Kosower, Edward M.; Newton, Gerald L.; Ranney, Helen M.

doi:10.1073/pnas.76.7.3382

Cited by 238 publications

(145 citation statements)

References 22 publications

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“…Cysl66 was selectively labelled with the fluorescent probe monobromotrimethyl-ammoniobimane as described earlier (Kosower et al, 1979;Picot et al, 1991). 1 mol trimethylammoniobirnane incorporated/mol monomer.…”

Section: Methodsmentioning

confidence: 99%

Crystal Structures and Solution Studies of Oxime Adducts of Mitochondrial Aspartate Aminotransferase

Marković-Housley

Schirmer

Hohenester

et al. 1996

European Journal of Biochemistry

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The interaction of mitochondrial aspartate aminotransferase with hydroxylamine and five derivatives (in which the hydroxyl hydrogen is replaced by the side chain of naturally occurring amino acids) was investigated by X-ray diffraction as well as by kinetic and spectral measurements with the enzyme in solution. The inhibitors react with pyridoxal 5'-phosphate in the enzyme active site, both in solution and in the crystalline state, in a reversible single-step reaction forming spectrally distinct oxime adducts. Dissociation constants determined in solution range from lo-' M to M depending on the nature of the side-chain group.The crystal structures of the adducts of mitochondrial aspartate aminotransferase with the monocarboxylic analogue of L-aspartate in the open and closed enzyme conformation were determined at 0.23-nm and 0.25-nm resolution, respectively. This inhibitor binds to both the open and closed crystal forms of the enzyme without disturbing the crystalline order. Small differences in the conformation of the cofactor pyridoxal phosphate were detected between the structures of both oxime complexes and the 2-methylaspartate adduct. The crystal structures indicate that the interaction between the w-carboxylate of the inhibitor and Arg292* of the neighbouring subunit is mainly responsible for the attainment of nearcoplanarity of the aldimine bond with the pyridine ring in the oxime adducts. Studies with a fluorescent probe aimed to detect shifts in the open/closed conformational equilibrium of the enzyme in oxime complexes showed that the hydroxylamine-derived inhibitors, even those containing a carboxylate group, do not induce the 'domain closure' in solution. This is probably due to the absence of the a-carboxylate group in the monocarboxylic hydroxylamine-derived inhibitors, emphasizing that both carboxylates of the substrates L -A s~ and L -G~ are essential for stabilizing the closed form of aspartate aminotransferase.

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Section: Methodsmentioning

confidence: 99%

Crystal Structures and Solution Studies of Oxime Adducts of Mitochondrial Aspartate Aminotransferase

Marković-Housley

Schirmer

Hohenester

et al. 1996

European Journal of Biochemistry

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“…The location of the thiols oxidized by the conditioned buffer or by H2O2 was determined by the administration of the fluorochromes monochlorobimane (20 M) or monobromotrimethylammoniobimane (20 M) (6,8). These compounds covalently react with reduced thiols, but if thiols are oxidized, the compounds do not bind.…”

Section: Animalmentioning

confidence: 99%

Redox-dependent coronary metabolic dilation

Saitoh¹,

Kiyooka²,

Ročić³

et al. 2007

American Journal of Physiology-Heart and Circulatory Physiology

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We have observed that hydrogen peroxide (H 2O2), the dismutated product of superoxide, is a coronary metabolic dilator and couples myocardial oxygen consumption to coronary blood flow. Because the chemical activity of H 2O2 favors its role as an oxidant, and thiol groups are susceptible to oxidation, we hypothesized that coronary metabolic dilation occurs via a redox mechanism involving thiol oxidation. To test this hypothesis, we studied the mechanisms of dilation of isolated coronary arterioles to metabolites released by metabolically active (paced at 400 min) isolated cardiac myocytes and directly compared these responses with authentic H2O2. Studies were performed under control conditions and using interventions designed to reduce oxidized thiols [0.1 M dithiothreitol (DTT) and 10 mM N-acetyl-L-cysteine (NAC)]. Aliquots of the conditioned buffer from paced myocytes produced vasodilation of isolated arterioles (peak response, 71% Ϯ 6% of maximal dilation), whereas H 2O2 produced complete dilation (92% Ϯ 7%). Dilation to either the conditioned buffer or to H 2O2 was significantly reduced by the administration of either NAC or DTT. The location of the thiols oxidized by the conditioned buffer or of H 2O2 was determined by the administration of the fluorochromes monochlorobimane (20 M) or monobromotrimethylammoniobimane (20 M), which covalently label the reduced total or extracellular-reduced thiols, respectively. H 2O2 or the conditioned buffer predominately oxidized intracellular thiols since the fluorescent signal from monochlorobimane was reduced more than that of monobromotrimethylammoniobimane. To determine whether one of the intracellular targets of thiol oxidation that leads to dilation is the redoxsensitive kinase p38 mitogen-activated protein (MAP) kinase, we evaluated dilation following the administration of the p38 inhibitor SB-203580 (10 M). The inhibition of p38 attenuated dilation to either H2O2 or to the conditioned buffer from stimulated myocytes by a similar degree, but SB-203580 did not attenuate dilation to nitroprusside. Western blot analysis for the activated form of p38 (phospho-p38) in the isolated aortae revealed robust activation of this enzyme by H2O2. Taken together, our results show that an active component of cardiac metabolic dilation, like that of H2O2, produces dilation by the oxidation of thiols, which are predominately intracellular and dependent activation on the p38 MAP kinase. Thus coronary metabolic dilation appears to be mediated by redox-dependent signals. coronary circulation; coronary microcirculation; reactive oxygen species; vasodilatation THE OXIDATION OF THIOL GROUPS is involved in many biological processes. Thiol oxidation induces protein conformation changes by converting the free thiols (-SH) into sulfenic acids (SO Ϫ ), sulfinic acids (SOO Ϫ ), sulfonic acids (SOOO Ϫ ), and disulfide bridges (S-S). Thiol oxidation is involved in many cellular processes, e.g., p38 mitogen-activating protein (MAP) kinase activation (2, 23), inhibition of p56 (lck) tyrosine kinase ...

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“…Our objective here was to viably and quantitatively separate recombinant GST' cells from the nonexpressing electroporated population by flow cytometry, and to determine the degree of cellular resistance to anti-BPDE conferred by expression of GST T. We sorted recombinant GST' cells on the basis of a GST-catalyzed intracellular conjugation of glutathione to monochlorobimane (mClB), a labeling reagent that i) is a GST substrate, and is nonfluorescent as a rcsctant and fluorescent once conjugated to glutathione (19,471; ii) is hydrophobic and rapidly diffuses across cellular membranes (28); and iii) specifically labels glutathione (45). Rice et al (45,471 analyzed cellular glutathione levels by flow cytometry using mClB, but sorting cells for levels of GST activity required us to equip a FACStarPLUS flow cytometer with a syringe pump to continuously infuse mClB on-line, ensuring that each cell was exposed to the substrate for a defined length of time before being sorted.…”

Section: Key Terms: Resistance To Carcinogens Reversion Of Transientmentioning

confidence: 99%

Recombinant glutathione S‐transferase (GST) expressing cells purified by flow cytometry on the basis of a GST‐catalyzed intracellular conjugation of glutathione to monochlorobimane

et al. 1991

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COS cells transiently expressing glutathione S-transferase (GST) m, Ya, or Yb, (human Pi, rat Alpha or Mu, cytosolic classes) were purified by flow cytometry and used in colony-forming assays to show that GST confers cellular resistance to the carcinogen benzoblpyrene (*)-anti-diol epoxide (anti-BPDE). We developed a sorting technique to viably separate recombinant GST+ cells (20%) from the nonexpressing electroporated population (80%) on the basis of a GST-catalyzed intracellular conjugation of glutathione to the fluorescent labeling reagent monochlorobimane (mClB). The concentration of mClB, length of time cells are exposed to mClB, and activity of the expressed GST isozyme determined the degree to which recombinant GST+ cells fluoresced more intensely than controls. On-line reagent addition ensured that all cells were exposed to 25 p M mClB for 3035 s during transit before being analyzed for fluorescence intensity and sorted. The apparent K , for mClB of the endogenous COS cell GST-catalyzed intracellular reaction was 88 pM. Stained GST Ya+ or Yb,+ cells catalyzed the conjugation 2 or 5 times more effectively than GST n+ cells. Enzyme activity in cytosolic fractions prepared from sorted recombinant GST+ cells was 1.8 -+ 0.3-fold greater than that of the control (80 f 4 nmoYmin/mg protein). Upon a 5-fold purification of GST n+ cells in the electroporated population, resistance to anti-BPDE in colony-forming assays increased 5 times, from 1.1-fold (unsorted) to 1.5-fold (sorted) (P <0.001).Key terms: Resistance to carcinogens, reversion of transient expression, COS cells Glutathione S-transferases (GST,3 EC 2.5.1.18) are a family of isozymes that can catalyze the covalent addition of the tripeptide glutathione, present at 0.5-10 mM in mammalian cells (35), to a structurally diverse array of physiological and xenobiotic electrophiles (21,25,32,37,41,52,53). GSTs are ubiquitous in the animal and plant kingdoms (53), and Mannervik (32) has classified the mammalian cytosolic GSTs into three classes (Pi, Alpha, and Mu) based on structural, immunological, and enzymatic properties. Mammalian cytosolic GSTs constitute 1-10% of total cytosolic protein (21) and are composed of two subunits (23-28 kilodaltons each) (32) that dimerize by noncovalent interactions (51). As demonstrated with in vitro kinetic analyses, GSTs can conjugate glutathione to anticancer alkylating agents (2,3,9,49,59) [1606][1607][1608][1609][1610][1611][1612] 1991) and Ublacker et al. (Cancer Res 51: 1783-1788, 1991, based upon in vitro kinetic analyses, support our findings done in whole cells which show that the rat )I and a class GSTs catalyze the conjugation of GSH to monochlorobimane more efficiently than 71 class GSTs.

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Bimane fluorescent labels: labeling of normal human red cells under physiological conditions.

Cited by 238 publications

References 22 publications

Crystal Structures and Solution Studies of Oxime Adducts of Mitochondrial Aspartate Aminotransferase

Crystal Structures and Solution Studies of Oxime Adducts of Mitochondrial Aspartate Aminotransferase

Redox-dependent coronary metabolic dilation

Recombinant glutathione S‐transferase (GST) expressing cells purified by flow cytometry on the basis of a GST‐catalyzed intracellular conjugation of glutathione to monochlorobimane

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