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PubMed1993

Interaction between red cell membrane band 3 and cytosolic carbonic anhydrase.

We have previously proposed that a membrane transport complex, centered on the human red cell anion transport protein, band 3, links the transport of anions, cations and glucose. Since band 3 is specialized for HCO3-/Cl- exchange, we thought there might also be a linkage with carbonic anhydrase (CA) which hydrates CO2 to HCO3-. CA is a cytosolic enzyme which is not present in the red cell membrane. The rate of reaction of CA with the fluorescent inhibitor, dansylsulfonamide (DNSA) can be measured by stopped-flow spectrofluorimetry and used to characterize the normal CA configuration. If a perturbation applied to a membrane protein alters DNSA/CA binding kinetics, we conclude that the perturbation has changed the CA configuration by either direct or allosteric means. Our experiments show that covalent reaction of the specific stilbene anion exchange inhibitor, DIDS, with the red cell membrane, significantly alters DNSA/CA binding kinetics. Another specific anion exchange inhibitor, benzene sulfonate (BSate), which has been shown to bind to the DIDS site causes a larger change in DNSA/CA binding kinetics; DIDS reverses the BSate effect. These experiments show that there is a linkage between band 3 and CA, consistent with CA interaction with the cytosolic pole of band 3.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1983

Microfluorometric studies on chromosomes. Quantitative determination of protein content of Chinese hamster chromosome 1 in situ with and without trypsin digestion.

The fluorescent stains, dansyl chloride and fluorescamine, were used to indicate the amount of protein in G-banded and unbanded chromosomes 1 of the Chinese hamster relative to the average amount of protein in human erythrocyte. Fluorescence was found to be proportional to protein mass up to the equivalent of three erythrocytes. In G-banding, trypsin digestion resulted in an average protein loss of 35.4% compared with unbanded chromosomes.

Animals↗
PubMed1975

The histochemical application of dansylhydrazine as a fluorescent labeling reagent for sialic acid in cellular glycoconjugates.

A new method for the fluorescent staining of stalic acid-containing glycoconjugates in fixed tissues is described. The procedure uses mild periodate oxidation, followed by condensation with dansylhydrazine and reduction of the hydrazones to hydrazines. The specificity of the reaction for sialic acid is tested on model glycoconjugates. The procedure gives superior resolution in comparison to the standard periodate Schiff procedure for cellular carbohydrates.

Animals↗
PubMed1976

The fluorescent demonstration of tissue aldehydes with dansylhydrazine.

Dansylhydrazine, previously introduced in a selective fluorescent cytochemical method for the demonstration of sialic acid residues of cellular glycoconjungates, may be broadly applied as a specific, covalently bonded fluorochrome of aldehyde residues, both those naturally occurring as in elastin or those generated through PAS or Feulgen-type procedures.

Aldehydes↗
PubMed1983

Enhanced membrane fluorescence of CDC-labelled paramecium subsequent to removal of surface components.

Cytofluorimetric analysis of cycloheptaamylose-dansyl chloride (CDC) labelled Paramecium indicates that after mild trypsin removal of surface components the localization of CDC on the outer surface of living cells was not modified by the treatment. After such treatment the intensity of fluorescence emission was found about 3-fold higher in treated single cell than in the untreated one. These findings indicate that CDC labelling can be used to follow alteration occurred on the membrane of the living cell prior to labelling.

Animals↗
PubMed1980

On the evaluation of photoreceptor properties by micro-fluorimetric measurements of fluorochrome diffusion.

By use of the microfluorimetric technique it is possible to study the diffusion of the fluorochrome di-dansylcystine (DDC) within isolated frog rod outer segments (ros) which are immobilysed in agarose gel. For this purpose, by a short hypotonic shock a leak is applied to one end of the ros. By this open end the DDC enters the rod and migrates through the whole outer segment. Following the propagation of the fluorescence boundary with time the cytoplasmatic diffusion constant can be determined if a chromatographic model is used to allow for the considerable binding of DDC to the inner membrane surface. With a binding constant K = 5 . 10(-4) cm the cytoplasmatic diffusion constant was found to be D = 1.3 . 10(-6) cm2/s whereas Dg = 2 . 10(-6 cm2/s and Dr = 3.5 . 10(-6) cm2/s were found in agarose gel or ringer solution, respectively. Using the mobility reduction factor given by D/Dr approximately equal to 0.4 to calculate the cytoplasmatic conductivity an inner resistance per length of 1.7 M omega/mu could be calculated for a frog rod which is in good agreement with corresponding data obtained from electrophysiological measurements.

Animals↗
PubMed1991

What does TnCDANZ fluorescence reveal about the thin filament state?

Fluorescence of skinned psoas fibres reconstituted with the troponin C subunit labelled with the fluorescent probe dansylaziridine (TnCDANZ) increases upon activation with Ca2+. This fluorescence enhancement is due to Ca2+ binding to the Ca(2+)-specific binding sites of TnCDANZ and attachment of cross-bridges to the actin filament. We found that approximately 20% of the enhanced fluorescence signal derived from Ca2+ binding to TnCDANZ and 80% from cross-bridge attachment during maximal activation. Furthermore we studied the effects of different cross-bridge states on TnCDANZ fluorescence. "Weakly" bound, non-force-generating cross-bridge states (pCa 8, low ionic strength) and rigor cross-bridges revealed similar effects on the TnCDANZ fluorescence. "Strongly" attached, force-generating states, however, increased fluorescence to the greatest extent. These results suggests a complex system of reciprocal couplings between TnC and different attached cross-bridge states. Cooling or increase of inorganic phosphate decreased isometric force but hardly decreased fluorescence, suggesting the accumulation of attached cross-bridge states with low tension output.

Actin Cytoskeleton↗
PubMed1992

Synthesis of methyl alpha- and beta-N-dansyl-D-galactosaminides, probes for the combining sites of N-acetyl-D-galactosamine-specific lectins.

The synthesis of the methyl alpha- and beta-N-dansyl-D-galactosaminides is described using methyl alpha,beta-2-azido-2-deoxy-D-galactopyranoside as starting material. This was reduced to the corresponding methyl alpha,beta-2-amino-2-deoxy-D-galactopyranoside and then treated with dansyl chloride to yield a mixture of methyl alpha,beta-N-dansyl-D-galactosaminides which was separated into individual anomeric forms by flash chromatography on silica gel. Methyl alpha-N-dansyl-D-galactosaminide was used as a fluorescent indicator ligand in continuous substitution titrations to determine the association constants of nonchromophoric carbohydrates with the N-acetyl-D-galactosamine specific lectin from Erythrina corallodendron.

Acetylgalactosamine↗
PubMed1980

Electrically induced release of amino acids formed from (U14C) glucose in rat brain cortex slices, studied by a simplified dansylation procedure.

The nonessential amino acids glutamate, aspartate, glutamine, gamma-aminobutyrate (GABA), alanine, glycine, and proline present in rat thin brain cortex slices were labeled by in vitro incubation of these with [U-14C]glucose, and the efflux of such endogenous radioactive amino acids and of lactate was studied in a superfused system, under control conditions or when the slices were depolarized by varous procedures. When electrical stimuli known to induce selective neurotransmitter release (1 or 1.5 volt, sine wave 60 Hz) were applied for 10 sec to the slices, no significant increase in amino acid efflux was found. When more intense stimuli (4 volt, 60 Hz) were applied for 60 sec, or extracellular potassium was raised to 56 mM, both conditions being known to induce nonselective substance release, the efflux of essentially all amino acids and of lactate was markedly increased. Increases in efflux were proportionately larger for glutamate, aspartate, and gamma-aminobutyrate, and this could be accounted for by their greater intracellular chemical (or electrochemical) potentials, but not because of a selective release mechanism for them. Amino acids were analyzed as their 1-dimethylaminoaphthalene-5-sulfonyl (dansyl) derivatives, by a modification of existing procedures in which the dansyl (DNS) derivatives were efficiently extracted from acidified incubation fluid into an organic phase. This rapidly desalted the derivatives and allowed their concentration and chromatographic separation on thin-layer silica gel sheets with little loss.

Amino Acids↗