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PubMed1983

Reversed phase thin-layer chromatography for the separation of beta-endorphin, beta-lipotropin and enkephalins.

A novel, simple technique using reversed phase thin-layer chromatography has been developed to separate beta-endorphin, beta-lipotropin and Met- and Leu-enkephalins. This method is useful for the rapid determination of the purity of these opioid peptides. The effectiveness of several solvent systems has been assessed. The resolution between beta-endorphin and beta-lipotropin achieved by this method exceeds that reported using high performance liquid chromatography or a variety of column techniques. This system also eliminates the problem of unrecognized loss of peptide due to binding on HPLC or other types of columns since the entire stationary phase is visualized using fluorescamine detection. Efficient, inexpensive and reliable simultaneous separation with visualization of several opioid peptides, their peptide precursors and their degradation products is now possible using the new technique. Such separations are required for many purposes including preparation of samples for radioimmunoassay of opioid peptides.

Chromatography, Thin Layer↗
PubMed1975

The conformational oscillation of delta-chymotrypsin involvement of methionine-192.

In delta-chymotrypsin the reactivity of methionine-192 towards p-nitrophenacyl bromide is strongly reduced when the alpha-amino group of isoleucine-16 has been acetylated. Since acetylation of isoleucine-16 brings delta-chymotrypsin to a conformation similar to its alkaline one this suggests that methionine-192 should present an impaired reactivity in the alkaline conformation of the protein. It is indeed observed that its chemical reactivity as a function of pH depends on the ionization state of the alpha-amino group of isoleucine-16 (pKapp 9 at 15 degrees C) as does the structure of the enzyme. Reciprocally, after chemical reaction of methionine-192 with hydrogen peroxide, isoleucine-16 presents a slower rate of reaction with fluorescamine than when methionine-192 is free. As a result of methionine-192 oxidation the apparent pK of the alkaline transition is shifted from 9 to about 11 at 15 degrees C. This is reflected in the disappearance of the lag phase previously observed for the initial activity of the enzyme when it is incubated at alkaline pH [Eur. J. Biochem. (1973) 39,293-300]. The absence of chemical reactivity of methionine-192 in the alkaline state of the enzyme is confirmed by the appearance of a lag phase in the reaction of the protein with iodoacetate after an incubation at alkaline pH. Such a lag phase does not appear when this incubation is carried out at neutral pH. Since this lag phase is similar to that which shows up in the activity during the isomerization of the enzyme from its alkaline to its neutral state, the present data are interpreted as implying a concerted movement of isoleucine-16 and methionine-192 during this isomerization process. They also indicate that in the alkaline form of the enzyme methionine-192 has moved back into the interior of the protein. Since the spectroscopic properties of the zymogen and of the high-pH form of the enzyme are similar they suggest that methionine-192 occupies in the alkaline conformation of the enzyme a similar position as it does in the zymogen.

Amino Acid Sequence↗
PubMed1977

Aminopeptidase N from Escherichia coli. Unusual interactions with the cell surface.

The subcellular localization of aminopeptidase N (previously called aminoendopeptidase) has been investigated. This enzyme was found to be partially released (30-40%) by osmotic shock or by converting Escherichia coli K10 cells to spheroplasts. However, in all other E. coli strains (K12, B/r, MRE 600, ML 308) tested, this enzyme is not released at all by these procedures and thus behaves like a cytoplasmic enzyme. The crypticity of aminopeptidase N is surprisingly low, 75-85% of the enzyme activity is directly assayable in intact cells of any E. coli strain. Various inhibitors of transport systems do not interfer with this assay. Aminopeptidase activity could also be assayed in spheroplasts, even when an insolubilized substrate was used, which suggests a surface location of this enzyme. As well, N-ethylmaleimide (0.4 mM), under conditions which do not allow penetration in the cytoplasm, caused 70% inhibition of aminopeptidase N. Binding of 125I-labeled antiaminopeptidase N antibody to spheroplasts (from K12 strain) was used to assay the orientation of aminopeptidase N in the membrane. This enzyme is exposed on the outer surface of the cytoplasmic membrane. Confirmation of this orientation was obtained by comparing the accessibility of aminopeptidase, alkaline phosphatase and beta-galactosidase to fluorescamine in intact cells. Only 16% of the total beta-galactosidase was labeled with this fluorescent reagent whereas 44-45% of the aminopeptidase N and 59% of the alkaline phosphatase were labeled. Electron microscopic visualization of insolubilized reaction products of aminopeptidase N within the cells showed that these products are located at the poles of the cells. Neither mutant cells which were devoid of aminopeptidase N activity nor parental strains with the enzyme activity inhibited with phenylmercuric chloride contained the characteristic black caps. Thus, it appears that the periplasm is enlarged at the poles of the cells and that the reaction product is mainly located in these places. Investigation of the type of interactions of aminopeptidase N with the plasma membrane only revealed that aminopeptidase N has mainly an electrostatic interaction with the outer surface, probably mediated by magnesium ion bridges. Additional interactions are involved since disruption of the integrity of the cytoplasmic membrane is required to totally release this enzyme.

Alkaline Phosphatase↗
PubMed1977

The isolation of tubulin and actin from mouse 3T3 cells transformed by simian virus 40 (SV3T3 cells), an established cell line growing in culture.

Tubulin can be purified from mouse SV3T3 cells (3T3 cells transformed by SV40 virus) by several cycles of temperature-dependent polymerization and depolymerization. Electron microscopical analysis of the final product reveals morphologically normal microtubules. Homogeneous actin can be isolated as a byproduct of the purification procedure. Mouse SV3T3 actin and skeletal muscle actin were compared by fingerprint analysis of the tryptic peptides obtained from performic-acid-oxidized protein. The two actins show a high degree of homology although apparently five of the twenty-five spots visualized by fluorescamine show a difference in chromatographic mobility. The purification procedure described allows the rapid isolation of both actin and tubulin from tissue culture in sufficient amounts for comparative biochemicals studies.

Actins↗
PubMed1991

Glycosphingolipids in insects. The amphoteric moiety, N-acetylglucosamine-linked phosphoethanolamine, distinguishes a group of ceramide oligosaccharides from the pupae of Calliphora vicina (Insecta: Diptera).

A group of Calliphora vicina pupal glycolipids could be segregated from the neutral glycosphingolipids, according to their two-dimensional TLC migration properties and positive reactions toward ninhydrin and fluorescamine spray reagents. These classified zwitterionic glycolipids were isolated by silica-gel column chromatography and characterized by the presence of a N-acetyl-glucosamine-bound phosphoethanolamine residue. The structural elucidation of the oligosaccharide moieties was performed by the determination of constituent carbohydrates as alditol acetates, linkage analysis by permethylation, exoglycosidase cleavage, fast-atom-bombardment mass spectrometry and NMR spectroscopy. The dominant fatty acid and sphingoid base species of the ceramide moieties were C20:0 (arachidic acid) and C14:1 (tetradecasphing-4-enine), respectively. The chemical structures of the zwitterionic, biogenetic glycosphingolipid series were determined as: (PEtn-6')GlcNAc(beta 1-3)Man(beta 1-4)Glc beta Cer; GalNAc(beta 1-4)(PEtn-6')GlcNAc(beta 1-3)Man(beta 1-4)Glc beta Cer; GalNAc(alpha 1-4)GalNAc(beta 1-4)(PEtn-6')GlcNAc(beta 1-3)Man(beta 1- 4)Glc beta Cer; Gal(beta 1-3)GalNAc(beta 1-4)(PEtn-6')GlcNAc(beta 1-3)Man(beta 1-4)Glc beta Cer; Gal(beta 1-3)GalNAc(alpha 1-4)GalNAc(beta 1-4)(PEtn-6')GlcNAc(beta 1- 3)Man(beta 1-4)Glc beta Cer; GlcNAc(beta 1-3)Gal(beta 1-3)GalNAc(alpha 1-4)GalNAc(beta 1-4)(PEtn- 6')GlcNAc(beta 1-3)Man(beta 1-4)Glc beta Cer.

Acetylglucosamine↗
PubMed1988

Reevaluation of taurine levels and distribution of cysteic acid decarboxylase in developing human fetal brain regions.

The possibilities of interference by glycerophosphoryl ethanolamine (GPE) in the estimation of taurine levels in cerebral cortex, midbrain, cerebellum, medullapons, and spinal cord of developing human fetal brain regions were eliminated by hydrolyzing tissue extracts with 6 M HCl. Cysteic acid thus produced was separated from taurine by ion-exchange chromatography using Biorad-AG resin. Fluorescamine was used as fluorogen. Data reveal that the estimation of taurine in human fetal brain regions is affected if GPE is present as a contaminant in the assay system. Cysteic acid decarboxylase activity was measured using cysteic acid as the substrate. Higher enzymic activity was recorded with increased fetal body weight, but the reverse was true for taurine level.

Body Weight↗
PubMed2006

Implications of sterol structure for membrane lipid composition, fluidity and phospholipid asymmetry in Saccharomyces cerevisiae.

Sterols are essential components of the plasma membrane in eukaryotic cells. Nystatin-resistant erg mutants were used in the present study to investigate the in vitro effects of altered sterol structure on membrane lipid composition, fluidity, and asymmetry of phospholipids. Quantitative analyses of the wild type and mutants erg2, erg3 and erg6 revealed that mutants have lower sterol (free)-to-phospholipid molar ratios than the wild type. Phosphatidylcholine content was decreased in erg2 and erg3 mutants; however, it was increased in erg6 strains as compared to normals. Phosphatidylserine content was increased in the erg6 mutant only. Fluorescence anisotropy decreased with temperature in both probes, and was lower for mutants than for the wild type, suggesting an increased freedom in rotational movement due to decreased membrane order. Investigation of changes in the aminophospholipid transbilayer distribution using two chemical probes, trinitrobenzene sulfonic acid and fluorescamine, revealed that the amounts of phosphatidylethanolamine derivatized by these probes were quite similar in both the wild type and various erg strains. The present findings suggest that adaptive responses in yeast cells with altered sterol structure are possibly manifested through changes in membrane lipid composition and fluidity, and not through transbilayer rearrangement of aminophospholipids.

Membrane Fluidity↗
PubMed1980

Purification of GABA on small columns of Dowex 50W; Combination with a method for separation of biogenic amines.

Elution of GABA has been included in the method for separation of biogenic amines on small Dowex 50W columns described by Atack & Magnusson (1978). After extraction with perchloric acid and neutralization to pH 3.0, the sample is passed through the column. 5-HIAA might thereafter be eluted as described by Lindqvist (1971). After washes with water and 8 ml of a 0.025 M sodium citrate buffer pH 4.5, GABA is eluted in 3 ml of a 0.05 M sodium citrate buffer pH 5.3-5.4. The elution of tryptophane and the above mentioned amines can thereafter be performed. The recovery of GABA from the column is 100%. Silica thin-layer chromatography of the GABA eluate gives only one ninhydrine positive spot with a Rf value identical to GABA standard. When the GABA eluate from a whole brain and spinal cord is run on an automatic amino acid analyser, only one peak is detectable, eluted in the same position as GABA standard. Fluorescence analysis of the GABA eluate gives similar values if a( ninhydrine, b) acetylacetone plus formaldehyde or c) fluorescamine is used to form the flurophore. The GABA values are identical whether the samples reacted with ninhydrine are diluted with water or copper tartrate. The GABA concentrations found in some parts of the rat brain are of the same order of magnitude as reported by other authors.

5-Hydroxytryptophan↗
PubMed1995

Molecular and physiological properties of clonidine-displacing substance.

An endogenous small molecular mass compound, termed clonidine-displacing substance (CDS), has been isolated and purified from bovine brain. The estimated level of CDS in bovine brain is 400-1,000 units/wet brain, with 1 unit of activity calculated to be approximately 1-2 ng. It is present in human serum, urine, and cerebrospinal fluid. The isolation procedure consists of initial aqueous and methanolic extractions followed by a series of HPLC chromatography steps (reverse phase and TSK sizing columns). The reverse-phase chromatography of CDS extracted under identical conditions from bovine brain and human serum show similar retention times. The final chromatography step gives a single active peak with a distinct ultraviolet spectrum, a single molecular peak m/z 587.8 +/- 2 in plasma desorption mass spectrometry (PDMS), and a unique pharmacological and physiological profile. Clonidine-displacing substance does not partition into organic solvents and it is ninhydrin and fluorescamine negative. All of these molecular properties clearly distinguish CDS from agmatine, an endogenous 130-dalton compound of far greater abundance which displays lower affinity for p-aminoclonidine-labeled sites in rat brain membranes. The ultraviolet spectrum of CDS consists of two aromatic peaks at 224 and 276 nm, whereas agmatine is an aliphatic substance with no ultraviolet absorbance. Like many antihypertensive drugs of the guanidine and imidazoline family of compounds, CDS recognizes alpha 2-adrenergic receptors, clonidine sites (IR-I1), and imidazoline sites (IR-I2). A good correlation exists between the affinities of various imidazoline/guanidine type ligands for IR-I2 in both human placenta and rat liver membranes which can be accurately determined because both tissues lack IR-I1 and alpha 2-adrenergic receptors. There is no correlation in the affinities of these ligands for IR-I2 of human-placental versus alpha 2-adrenergic receptors of human platelets. By uncovering the role of CDS in the central nervous system we will be able to understand the coupling of IRs to neurotransmission and, in turn, to changes in arterial pressure.

Agmatine↗
PubMed1985

Comparative aspects of circulating erythrocytes in the trophic and reproductive phases of European eel. Ultrastructure and cytochemistry.

Different cytochemical and morphological parameters were used to compare the functional patterns of the erythrocytes in the transition from the trophic (yellow eel) to the reproductive (silver eel) phases of European eel (Anguilla anguilla L.). Data on the nucleus were obtained by microdensitometric (Feulgen reaction) and cytofluoremetric (propidium iodide and Hoechst 33342 fluorochromization) evaluations of DNA staining intensity. In the silver eel, chromatin condensation was more heterogeneous and chromatin also appeared to be looser. The fluorochromes provided information on the different degrees of organization of the chromatin filament, with regard to both primary and higher order DNA structure. In the silver eel, the fine structure showed a higher number of erythrocytes with large euchromatinic areas and the heterochromatinic component was occasionally present in scattered clumps. As for the cytoplasm, in the silver eel a positive correlation could be established between the presence of intact cytoplasmic organelles (mitochondria, Golgi complexes, ribosomes, etc.) and the high fluorescence intensity. This aspect is revealed through the detection of definite membrane components such as glycoconjugates and primary amino groups (PAS reaction and fluorescamine staining, respectively). All these findings allowed us to establish that the transition from the trophic to the premigratory reproductive phase is marked by a higher heterogeneity of the erythrocytes, due to different cell maturation levels. This fact can be ascribed to different metabolic requirements as well as to a more intense erythropoiesis, typical of the most active phase of the eel's life.

Aging↗
PubMed1990

Cephalosporin C acylase in the autolysis of filamentous fungi.

Cephalosporin C acylase activity was studied using fluorescamine determination of free--NH2 groups produced in the deacylation of cephalosporin C by the enzyme. Fourteen fungi from different genera were studied and low extracellular cephalosporin C acylase activity was found in the genera Aspergillus, Fusarium and Penicillium. Forty one fungi of these genera were checked but not all presented acylase activity. The enzyme was generally found to be an extracellular enzyme and during the process of autolysis its activity increased with incubation time and with increasing pH of the medium. In no case was beta-lactamase activity detected. Penicillium rugulosum and Penicillium griseofulvum were identified as good cephalosporin C acylase producers. Deacetyl esterase activity was also detected in these fungi.

Amidohydrolases↗
PubMed1987

Determination of teicoplanin concentrations in serum by high-pressure liquid chromatography.

An isocratic reversed-phase high-pressure liquid chromatographic method for the determination of six components of the teicoplanin complex in biological fluid was developed. By using fluorescence detection after precolumn derivatization with fluorescamine, the assay is specific and highly sensitive, with reproducibility studies yielding coefficients of variation ranging from 1.5 to 8.5% (at 5 to 80 micrograms/ml). Response was linear from 2.5 to 80 micrograms/ml (r = 0.999); the recovery from spiked human serum was 76%. An external quality control was performed to compare this high-pressure liquid chromatographic method (H) with a standard microbiological assay (M); no significant deviation from slope = 1 and intercept = 0 was found by regression analysis (H = 1.03M - 0.45; n = 15).

Anti-Bacterial Agents↗
PubMed1979

Antigenic polypeptide complex from the Melvin strain of Neisseria gonorrhoeae: isolation and properties.

An antigenic complex has been isolated in a highly purified from from the Melvin strain of Neisseria gonorrhoeae. The complex has a molecular weight of 9.3 x 10(6) and on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was found to consist of several subunits; the most predominant had the following molecular weights: 110,000, 94,000, 68,000, a smear containing (52,000, 48,000, and 44,000), 42,000, 36,000, 29,000, 28,000, 26,000, and 12,000 comprising 89% of the total protein. With the exception of the subunit of molecular weight 110,000, no change in the content or the mobility of other subunits was observed when beta-mercaptoethanol was omitted from the denaturation solution of sodium dodecyl sulfate electrophoresis. Amino acid analysis of the complex showed a predominance of hydrophobic amino acids. These data implicated noncovalent interactions between the subunits. When the cells were labeled with fluorescamine it was possible to obtain a fluorescent complex with identical properties. Among several buffers used for the isolation of the complex, 0.2 M tris(hydroxymethyl)aminomethane buffer (pH 7.5) gave maximum yield with low amounts of lipopolysaccharide and phospholipid; the choice of the buffer for column chromatography did not seem to make any difference. The high protein content and low amounts of lipopolysaccharide and phospholipid are characteristic properties of the complex.

Amino Acids↗
PubMed1979

Isolation and characterization of toxic fractions from Brucella abortus.

Two types of toxic fractions, protein-rich and carbohydrate-rich, were isolated from attenuated (strain 19) and virulent (strain 2308) Brucella abortus organisms. Polyacrylamide gel electrophoresis of the protein-rich fraction, in the presence and absence of sodium dodecyl sulfate, revealed qualitative and quantitative differences in the protein bands derived from the attenuated and virulent strains. Sodium dodecyl sulfate-gel electrophoresis indicated that the major differences between these protein fractions were in the molecular weight range from 14,000 to 40,000. Immunoelectrophoresis of these fractions from the attenuated and virulent strains revealed differences in the antigenic spectrum. Polypeptides in the carbohydrate-rich fraction could be visualized on polyacrylamide gels only when reacted with fluorescamine before electrophoresis. Immune sera did not precipitate the components of the carbohydrate-rich fraction. Intradermal injecttion of the protein and carbohydrate-rich fractions resulted in different types of skin lesions in guinea pigs, i.e., edematous/erythematous and necrotic lesions, respectively. Fractions derived from attenuated and virulent strains of B. abortus were equally toxic in the guinea pig skin test. The toxic activity of both types of fractions was susceptible to pronase and heat treatment.

Animals↗
PubMed1984

Qualitative detection of muramic acid in normal mammalian tissues.

Rat tissues were extracted with 8% trichloroacetic acid, and the products were hydrolyzed with hydrochloric acid and purified. Fluorescamine derivatives were made and subjected to thin-layer chromatography; material with an Rf corresponding to the authentic muramyl derivative was obtained. It was oxidized with periodate, and the resulting formaldehyde was identified fluorimetrically. Alternatively, treatment with base released D-lactate (beta-elimination), which was identified fluorimetrically by reduction of NAD to NADH with D-lactate dehydrogenase. The data indicate that small muramyl compounds, presumably peptides of bacterial origin, are normally present in rat liver, brain, and kidney. The functions of muramyl compounds are suggested by much recent work.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed1991

Inhibition of bactericidal and bacteriolytic activities of poly-D-lysine and lysozyme by chitotriose and ferric iron.

In a previous report from this laboratory (N. J. Laible and G. R. Germaine, Infect. Immun. 48:720-728, 1985), evidence was presented to suggest that the bactericidal actions of both reduced (i.e., muramidase-inactive) human placental lysozyme and the synthetic cationic homopolymer poly-D-lysine involved the activation of a bacterial endogenous activity that was inhibitable by N,N',N"-triacetylchitotriose (chitotriose). In the present investigation however, we found that the bactericidal and bacteriolytic action of poly-D-lysine could be prevented only by some commercially available chitotriose preparations and not by others. Analysis by physical and chemical methods failed to distinguish protective chitotriose (CTa) and nonprotective chitotriose (CTi) preparations. CTi and CTa preparations displayed equal capacities to competitively inhibit binding of [3H]chitotriose by immobilized lysozyme and were indistinguishable in their abilities to block the lytic activity of lysozyme against Micrococcus lysodeikticus cells. Elemental analysis revealed significantly higher levels of phosphorus, calcium, iron, sodium, manganese, and copper in CTa. Removal of metals from CTa by chelate chromatography completely abolished the poly-D-lysine-protective capacity. Of the metals detected, only ferric iron (5 to 10 microM) mimicked the protective action of CTa. A Fe(III) concentration of 50 microM was required to inhibit lysozyme (5 micrograms/ml). Both Fe(III) and CTa (but not CTi) quantitatively blocked the labeling of poly-D-lysine by fluorescamine, suggesting that the primary amino groups of the lysine residues participate in iron binding. Thus, it appears that the poly-D-lysine-protective capacity of certain chitotriose preparations was due not to the chitotriose itself but to contaminating metal ions which interact directly with the polycationic agent. In contrast, Fe(III) cannot account for inhibition of either the bactericidal or bacteriolytic activity of lysozyme by chitotriose.

Bacteria↗
PubMed1978

Structural components of mouse mammary tumor virus. II. Isolation and purification of virion polypeptides.

Mouse mammary tumor virus (MMTV) glycoproteins and nonglycosylated polypeptides were purified by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Primary amino groups were labeled with fluorescamine to enable visualization of MMTV polypeptides in the gels. Protein bands were sliced from the gels and eluted with 90 to 95% recovery. Eight MMTV polypeptides, including three of the major viral components as well as five minor proteins, were routinely obtained. Double diffusion assays and immunoelectrophoresis confirmed the retention of antigenicity identical to that of untreated MMTV virions. Antisera obtained from MMTV-free BALB/c mice immunized with these purified proteins reacted with the polypeptide immunogen as well as with detergent-disrupted MMTV virions from mouse milk or cell culture. Double diffusion assays using the specific mouse antisera failed to detect any cross-reactivity among the isolated polypeptides. A hemagglutination-inhibition assay demonstrated that the ability of MMTV virions to inhibit the hemagglutinating properties of influenza virus resides in the glycosylated polypeptides gp52, gp37.7, and gp33.

Antigens, Viral↗
PubMed1986

Studies of the free faecal amines of infants with gastroenteritis and of healthy infants.

The free primary amines present in the faeces of 44 infants (1-18 months) with gastroenteritis have been examined by field desorption mass spectrometry of the lactone form of their fluorescamine derivatives without their prior separation. p-Tyramine, 2-phenylethylamine, the diamines, putrescine and cadaverine and several of their acyl derivatives were common constituents, but a number of other amines were also characterised. Using thin layer chromatography and field desorption mass spectrometry of the amine dansyl derivatives a comparative study was made of the faecal amines of 13 selected infants with gastroenteritis (diarrhoea) and of 13 healthy infants. p-Tyramine, the most abundant amine, was significantly higher (p = 0.02) in the sick infants. The overall presence of p-tyramine was more significantly related to the diet of the infants. Faecal tyramine was low in breast fed infants but significantly higher (p = 0.01) in infants fed cow's milk.

Amines↗