Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Fluorescamine”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 613 records · Page 34Linked to original sources
PubMed1988

An endosomal model for acid triggering of diphtheria toxin translocation.

An endosomal model system was developed for studying the effects of pH on vesicle-entrapped diphtheria toxin. The "endosomes" were prepared from dioleoylphosphatidylcholine (1 mg), diphtheria toxin (0.25 mg), and lysozyme (2.25 mg) in water at pH 8.4. The method used for preparing large unilamellar vesicles was adapted from the procedure of Shew and Deamer (Shew, R. L., and Deamer, D. W. (1985) Biochim. Biophys. Acta 816, 1-8). Efficiencies of trapping (typically 45-75%) and separation from untrapped proteins (typically 95-100%) were assessed by fluorescamine assays conducted before and after column chromatography and in the presence and absence of Tergitol Nonidet P-40. Intramembranous photolabeling revealed that diphtheria toxin inserts into the vesicle bilayer when the pH is dropped to 4; surface labeling revealed that the same treatment leads to exposure of diphtheria toxin at the trans surface of the vesicles. Release of toxin to the solution was not detected under the experimental conditions employed (i.e. with nicked or unnicked toxin, +/- exogenous trypsin, pH 4 or 8.4). Preliminary results indicate that this model system will be a valuable tool for elucidating the pathway by which the ADP ribosyltransferase domain of diphtheria toxin gains access to the cytoplasmic compartment of cells after endosomal uptake.

ADP Ribose Transferases↗
PubMed1986

The amino acid sequence of a delta 5-3-oxosteroid isomerase from Pseudomonas putida biotype B.

We have determined the primary structure of a delta 5-3-oxosteroid isomerase from Pseudomonas putida biotype B. The enzyme is a dimeric protein of two identical subunits, each consisting of a polypeptide chain of 131 residues and a Mr = 14,536. The intact S-carboxymethyl protein was sequenced from the NH2 terminus using standard automated Edman degradation and automated Edman degradation using fluorescamine treatment at known prolines to suppress background. The isomerase was fragmented using CNBr, trypsin, iodosobenzoic acid, and acid cleavage at aspartyl-prolyl peptide bonds. The peptides resulting from each fragmentation were separated by reversed-phase high performance liquid chromatography and sequenced by automated Edman degradation. The full sequence was deduced by the overlapping of the various peptides. A search for homologous proteins was performed. Only the oxosteroid isomerase from Pseudomonas testosteroni, an expected homology, was found to be similar. Comparison of the two proteins shows that the region of strongest homology is the region containing the aspartic acid at which steroidal affinity and photoaffinity reagents have been shown to react in the P. testosteroni isomerase. The P. putida isomerase contains 3 cysteines and 2 tryptophans, whereas the P. testosteroni isomerase lacks these amino acids. The two proteins are not highly conserved.

Amino Acid Sequence↗
PubMed1986

Localization of phosphatidylethanolamine in microsomal membranes and regulation of its distribution by the fatty acid composition.

Rat liver microsomes were incubated with the monofunctional aminoreagent fluorescamine. Although the probe easily penetrated the membranes, two pools of phosphatidylethanolamine (PE) could be detected. The first pool rapidly reacted with the probe and comprised 80% of the total PE. The second pool exhibited a very slow interaction. The two pools showed differences in fatty acid composition as well as in their sites of attachment. In vivo labeling with ethanolamine, glycerol, and palmitic and stearic acid resulted in a higher specific activity in the first pool after 1 hr; equilibration with the second pool took about 3 hr. No equilibration between the pools could be detected under in vitro conditions. In vivo incorporation of labeled fatty acids showed that palmitic and stearic acids were mainly incorporated into phosphatidylethanolamine by de novo synthesis, while linoleic and arachidonic acids were introduced through deacylation-reacylation processes. Injection of liposomes consisting of labeled synthetic phosphatidylethanolamines into the portal vein was followed by uptake by the hepatocytes and incorporation of the lipids into the microsomal membranes. Depending on the fatty acid composition of the injected lipid, one of either of the two pools became labeled. It is suggested that the fatty acid composition of a given phospholipid molecule exerts a signal function directing the lipid to its final intramembranous location.

Animals↗
PubMed1979

Liquid-chromatographic measurement of taurine in cerebrospinal fluid of normal individuals and patients with meningitis.

Taurine was measured in cerebrospinal fluid by reacting it with fluorescamine to form a fluorescent derivative, followed by separation on a reversed-phase column and fluorometric detection and evaluation. The assay is rapid (17 min) and sensitive to as little as 1 mumol/L. The mean value for 27 cerebrospinal fluid samples collected from patients free from meningitis and aneurysm was 5.7 +/- 1.8 mumol/L. Twenty-two patients with bacterial meningitis showed a 0- to 20-fold increase in cerebrospinal fluid taurine, with a return to normal values after antibiotic treatment.

Chromatography, High Pressure Liquid↗
PubMed1978

Fluorescent analogue of ampicillin.

As the outcome of the reaction of ampicillin with 4-phenylspiro[furan-2(3H),1'-phthalane]-3,3'-dione (fluorescamine), the chemical analogue of ampicillin (F-AMPI) has been prepared. This preparation has manifested strong fluorescence in UV light. Some basic chemical and biological properties of this product such as chemical stability electrophoretic mobility, uniformity during thin-layer chromatography and ion-exchange chromatography, and antimicrobial activity were examined. The results were compared with values of the original antibiotic.

Ampicillin↗
PubMed1980

The common peptides and the cytochemistry of their cells of origin.

Thirtyfive biologically active peptides are products of the 40 cells of the APUD series, which constitute the Diffuse Neuroendocrine System (DNES). Twentyone of these peptides are found not only in the cells and processes of the DNES but also in the cells and the processes of the nervous system. Hence the appellation common. To the seven original common cytochemical features of the APUD cells it is possible to add the positive results of 3 subsidiary staining techniques (Lead haematoxylin, Argyrophilia, Formaldehyde-Fluorescamine), a singly cytochemical method (Formaldehyde-ozone), and an immunocytochemical molecular marker method (neuron-specific enolase; NSE). Application of this last method demonstrates NSE in the APUD cells and confirms their origin from "neuroendocrine-programmed epiblast."

APUD Cells↗
PubMed1983

The binding properties of human complement component C1q. Interaction with mucopolysaccharides.

Quantitative measurements have been made of the interaction of human complement subcomponent C1q with mucopolysaccharides. The binding of C1q to heparin was quantitatively examined by utilizing an assay that employs a 125I-labeled low molecular weight heparin glycosaminoglycan (LMW-Hep) (Mr = 8500). Two classes of binding sites were detected. The first class of sites bound 2.02 mol of LMW-Hep/mol of C1q with a Kd of 76.6 nM. The second class of sites complexes with 12 mol of LMW-Hep with a Kd of 1.01 microM. The higher affinity-binding site for LMW-Hep could be assigned to the collagenous region of C1q (C1q-c); 2.2 mol of 125I-LMW-Hep were bound/mol of purified isolated C1q-c with a Kd = 381 nM. In contrast, the isolated C1q globular region did not bind to 125I-LMW-Hep. The binding of LMW-Hep to C1q and the C1q-c region was confirmed by fluorescence polarization experiments; C1q and C1q-c bound 2.3 and 2.02 mol of fluorescamine-labeled LMW-Hep/mol of protein, respectively. A variety of mucopolysaccharides were able to inhibit interaction of C1q with 125I-LMW-Hep, the most effective being heparan sulfate and dermatan sulfate. LMW-Hep (2.5 nM) inhibited the ability of C1q (0.5 nM) to recombine with C1r (1.4 nM) and C1s (1.6 nM) to form hemolytically active C1. At 250 mM, LMW-Hep inhibited the hemolytic activity of reconstituted C1. The ability of mucopolysaccharides to interact with purified C1q suggests a role for such molecules in the regulation of the first component of complement.

Centrifugation, Density Gradient↗
PubMed1981

Purification and characterization of the 53,000-dalton glycoprotein from the sarcoplasmic reticulum.

A 53,000-dalton intrinsic glycoprotein of the sarcoplasmic reticulum was separated from the Ca2+ + Mg2+-ATPase by dissolution with low concentrations of deoxycholate in the presence of 1 M KCl and purified in two successive gel filtration steps. It was aggregated and eluted at the void volume when subjected to gel filtration in the presence or absence of deoxycholate. When subsequently chromatographed in the presence of sodium dodecyl sulfate, the glycoprotein eluted in pure form as a monomer. The glycoprotein contained 48% nonpolar amino acids. It also contained 4 mol of glucosamine and 18 mol of mannose per mol of protein, suggesting that it contained two chains of (GlcNAc)2, (Man)9. The 53,000-dalton glycoprotein was completely removed from deoxycholate extracts of sarcoplasmic reticulum by affinity chromatography on concanavalin A Sepharose. Elution of glycoproteins with alpha-methyl-D-mannoside and deoxycholate resulted in co-purification of the 53,000-dalton glycoprotein and 160,000-dalton glycoprotein previously observed in sarcoplasmic reticulum. The apparent molecular weight of the glycoprotein was reduced from 53,000 to 49,000 after digestion with endo-beta-N-acetylglucosaminidase H (Endo H) and its reactivity with concanavalin A (Con A) was lost. There was no change in molecular weight of the glycoprotein and no diminution of its reactivity with Con A when sealed vesicles of sarcoplasmic reticulum were treated with Endo H. Endo H reduced the molecular weight and the Con A reactivity of the protein when the vesicles were made permeable by detergents. These observations, together with our previous demonstration that the glycoprotein reacts with a cycloheptaamylose-fluorescamine complex in sealed vesicles (Michalak, M., Campbell, K. P., and MacLennan, D. H. (1980) J. Biol. Chem. 255, 1317-1326), show that the glycoprotein is a transmembrane protein. A protein of approximately 53,000 daltons was labeled when the sarcoplasmic reticulum was reacted with the photoaffinity label [32P]8-N3-cAMP. The labeled protein was neither the glycoprotein nor the high affinity calcium-binding protein since it was not sensitive to Endo H and was sensitive to trypsin digestion.

Affinity Labels↗
PubMed1984

Simplified fluorometric assay for sphingosine bases.

A simplified procedure for fluorometric determination of sphingosine bases is presented. Nitrogenous bases are reacted with fluorescamine and then extracted into a lower phase solvent, obviating the need to transfer the reaction products prior to quantitation. Consequently, the entire procedure may be carried out in a single set of screw-capped reaction tubes. Chloroform was found to be the best solvent of those tested for the maximal extraction of derivatized sphingosine bases with negligible extraction of nitrogenous contaminants derived from phospholipids and amino sugars. The simplified method retains excellent sensitivity and selectivity and allows for simultaneous processing of large numbers of samples. Furthermore, quantitation of glycolipid-derived hexosamine can be obtained by measuring the fluorescence of the aqueous phase after partitioning.

Chloroform↗
PubMed1978

Thin layer chromatographic screening method for sulfadiazine residues in calf tissues, plasma, and urine.

Because of the lack of specificity of the Bratton-Marshall procedure for assaying sulfonamides, a sensitive, specific tissue residue assay for sulfadiazine (SDZ) was developed. The methodology has been extended to provide a highly sensitive screen for sulfonamide residues, which employs 2-dimensional thin layer chromatography in conjunction with fluorescamine derivatization. The procedure described, which has been developed for SDZ in calf tissues, involves direct ethyl acetate extraction of tissue homogenates. Following evaporation of the organic phase, a portion of the residue is spotted on a 20 X 20 cm silica gel 60 plate, which is then developed in 2 dimensions with solvent systems devised to separate SDZ from endogenous substances as well as from 12 other sulfonamides that might be present in calf tissues. The presence of SDZ at a concentration of 0.1 ppm or its absence is easily demonstrated in calf kidney, liver, muscle, plasma, and urine. The basic method can be modified for a particular sulfonamide in a target tissue and can be used as a quantitative assay for sulfonamide residues.

Animals↗
PubMed1984

[Microassay of epsilon-aminocaproic acid by liquid chromatography].

A method for the analysis of epsilon aminocaproic acid in plasma or serum has been developed. This report describes a simple and rapid assay with a small sample (20 microliter) by reverse phase high performance liquid chromatography. The supernatant is derivatized with fluorescamine after acetonitrile deproteinisation. Linearity, sensibility and accuracy are good. Save time extraction allows quick determination of many samples.

Aminocaproates↗
PubMed1984

Direct determination of human and rabbit apolipoprotein B selectively precipitated with butanol-isopropyl ether.

A method is described for the rapid, selective, and quantitative precipitation of apolipoprotein B from isolated hypercholesterolemic rabbit and human very low density lipoproteins (VLDL), intermediate density lipoproteins (IDL), and low density lipoproteins (LDL). Lipoprotein samples are heat-treated at 100 degrees C in 1% SDS. The denatured apoprotein solutions are then mixed briefly with two volumes of butanol-isopropyl ether 45:55 (v/v) to precipitate the apoB. The supernatant solutions, containing the non-apoB proteins and lipids, are removed and the apoB pellet is washed once with water. To determine apoB specific activity, the apoB pellet is resolubilized in 0.5 M NaOH by heating for 30 min at 120 degrees C. The hydrolyzed apoB protein is quantitated by fluorescence of a fluorescamine derivative. The precipitation of apoB is quantitative and selective: 99.5% of rabbit 125I-labeled LDL-apoB and 97.5% of human 125I-labeled LDL-apoB is precipitated and less than 5% of 125I-labeled HDL added to unlabeled VLDL, IDL, or LDL is precipitated. Triglyceride and cholesteryl ester contamination of the apoB pellet is less than 2% of their original radioactivities.

Animals↗
PubMedDate not supplied

Fluorometric estimation of taurine in tissue extracts and biological fluids.

A fluorometric technique was developed to estimate taurine in tissue extracts and body fluids. Dowex-AG and Biorad-AG columns were used to separate this amino acid from other components. Interference by Glycerophosphoryl ethanolamine was removed by hydrolysis of the sample with 6N HCl. The fluorogen used was fluorescamine.

Animals↗
PubMedDate not supplied

[Spectrofluorimetric procedures for estimation of lysosomal cathepsins A, B C and D activities].

Unified modifications of procedures are described for estimation of lysosomal cathepsins A, B, C and D activities. The procedures are based on spectrofluorimetric determination of beta-naphthylamine in the experiments with cathepsins B and C or on spectrofluorimetric determination of fluorescamine conjugates with epsilon-NH2-groups of free amino acids in the case of cathepsins A and D. Kinetics of all the reactions studied obeyed the Michaelis-Menten equation; the enzymatic activities could be estimated at protein concentration up to 0.1 mg/ml of the experimental sample.

Animals↗
PubMed1983

Quantitative thin layer chromatographic multi-sulfonamide screening procedure.

In-situ optical scanning of fluorescamine derivatives on thin layer silica gel plates provides a rapid method for the determination of multiple sulfonamides at levels below 0.1 ppm. Sample preparation is minimal. Homogenized liver or muscle is extracted with ethyl acetate and then back-extracted into 0.2M glycine buffer. After pH adjustment, the extract is washed with hexane and extracted with methylene chloride. The organic phase is evaporated to dryness and reconstituted in methanol. Pre-adsorbent layer silica gel plates are used for chromatography. The method has been applied to residues of sulfamethazine, sulfadimethoxine, sulfathiazole, sulfaquinoxaline, and sulfabromomethazine in cattle, swine, turkey, and duck tissues.

Animals↗
PubMed1982

Subcellular distribution of particle-associated antigens in Trypanosoma rhodesiense.

Bloodstream forms of the African trypanosome Trypanosoma rhodesiense were subjected to analytic cell fractionation procedures using isopycnic sucrose gradient centrifugation. The fractions obtained were analyzed using fused rocket immunoelectrophoresis, which revealed the most antigenic components to be associated with particles sedimenting between density increments of 1.10 to 1.15 and 1.20 and 1.22. From the distribution of various marker enzymes and the fluorogenic label fluorescamine, these particle populations were identified as derived from flagella pocket and surface membrane, respectively. Crossed immunoelectrophoresis of postulated flagella pocket and surface membrane fractions demonstrated at least seven and 10 distinct antigens, respectively, with only limited cross-reactivity. On the basis of crossed affini-immunoelectrophoresis four to five of each of these membrane antigens were glycoproteins, being precipitated in a concanavalin A-containing gel. Of the various detergent treatments used, it was found that optimum extraction of these glycoprotein antigens was achieved using the zwitterionic detergent Zwittergent 3-12 (0.1%) in the presence of 0.4% Triton X-100. Fused rocket immunoelectrophoresis also revealed a single prominent antigen associated with a particle population having an equilibrium density of approximately 1.180, which was identified as lysosomal from the distribution of marker enzymes. Other intracellular sites were not significantly antigenic.

Acid Phosphatase↗
PubMed1977

Analysis of soluble proteins in comedones.

The detergent-soluble fraction of purified, concentrated comedo samples was analysed for protein by disc-gel electrophoresis and fluorescamine assay. Spectrophotometer scanning revealed a characteristic pattern of 14 peaks which was essentially uniform in comedones from individuals having acne, Favre-Racouchot syndrome, or prepubertal comedones. The pattern was different in material derived from keratinous cysts and the contents of follicles from the nose. The presence of small amounts of serum proteins, notably globulins, was detected by immunodiffusion.

Acne Vulgaris↗
PubMed1981

Identification of a 2-aminotetrose in a lipid-soluble fraction of rat liver.

Chromatography on DEAE-cellulose of chloroform:methanol:water (10:10:3) extracts obtained from the livers of rats pulsed 50 min with [32P]Pi and D-[1-3H]-mannose yielded two radioactive components which eluted at salt concentrations greater than 0.1 M. One of the components yielded, upon mild acid treatment (20 mM HCl, 100 degrees C, 20 min), a low molecular weight water-soluble component (PO), which contained most of the 32P label associated with the parental lipid-oligosaccharide. PO was periodate-negative, ninhydrin-negative, and permanganate-positive. The phosphate group was stable to strong acid, which converted PO to a single, 32P-labeled product, but the phosphate was readily released by incubation under mild alkaline conditions. Strong alkali produced a product reactive with periodate, ninhydrin, and fluorescamine. When dephosphorylated PO was hydrolyzed with acid and the products were converted to the alditol acetate derivatives, gas chromatography revealed a single major component. Gas chromatography/mass spectroscopy of this compound showed it to be the derivative of a 2-acetamido-2-deoxytetrose. That 2-acetamido-2-deoxytetrose is glycosidically bound in PO is indicated by the fact that PO must be hydrolyzed by strong acid before reduction with NaBD4 is able to incorporate deuterium into the tetrose.

Amino Sugars↗