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B Colas

Publications and source records attributed to B Colas.

53 records · Page 3Linked to original sources
PubMed1987

Soluble form of acetylcholinesterase from rabbit enterocytes: comparison of its molecular properties with those of the plasma membrane species.

A soluble form of acetylcholinesterase was shown to be present in rabbit enterocytes. The enzyme was obtained from a high-speed supernatant (105,000 X g centrifugation) after homogenization of intestinal mucosa without detergent. It was shown to possess no obvious hydrophobic character and could be classified as a low-salt-soluble (LSS) acetylcholinesterase. Sucrose gradient centrifugation revealed a single enzyme species with a sedimentation coefficient of 3.9 +/- 0.2S. By gel filtration performed in HPLC the enzyme was eluted as a protein corresponding to an Mr of 72,000 +/- 3,000. It could be precipitated with concanavalin A by affinoelectrophoresis, but the catalytic activity was not affected by the lectin. Our results are consistent with a G1 globular form for this soluble acetylcholinesterase which differs very clearly from detergent-soluble forms also found recently in the plasma membranes of rabbit enterocytes.

Acetylcholinesterase↗
PubMed1985

Presence and characterization of acetylcholinesterase in brush-border and basolateral membranes of rabbit enterocytes.

Acetylcholinesterase is found in the brush-border and basolateral membranes purified from rabbit enterocytes. The sedimentation coefficients of the enzymes solubilized from two types of membrane are identical (5.5 +/- 0.2 S) and the apparent molecular weights are not significantly different (154 000 +/- 8000 for the brush-border and 145 000 +/- 8000 for the basolateral membrane enzyme). These results suggest a unique G2 molecular form for acetylcholinesterase from brush-border as well as from basolateral membranes.

Acetylcholinesterase↗
PubMed1983

Glycosylation of yeast aspartyl-tRNA synthetase. Affinity labelling by glucose and glucose 6-phosphate.

Several lines of evidence establish that the crystallizable aspartyl-tRNA synthetase from Baker's yeast contains some covalently bound glucose: (i) a positive staining of the enzyme was obtained after polyacrylamide gel electrophoresis followed by the concanavalin A-peroxidase test which is specific for glucose and mannose containing proteins; (ii) thin-layer chromatography and gas-liquid chromatography revealed the presence of glucose in enzyme hydrolysates; (iii) immunoaffinoelectrophoresis in agarose gels containing concanavalin A and antibodies raised against aspartyl-tRNA synthetase showed that the enzyme was able to precipitate entirely in the lectin. Finally incubation of the enzyme with [14C]glucose or [14C]glucose 6-phosphate led to the incorporation of radioactivity into trichloroacetic acid-precipitable protein. Indeed immunoprecipitation of [14C]glucose-labelled aspartyl-tRNA synthetase with specific antibodies using the rocket method followed by autoradiography gave a radioactive peak. This last result also demonstrates the possibility of in vitro glycosylation of yeast aspartyl-tRNA synthetase.

Affinity Labels↗
PubMed1983

Phaseolus vulgaris cytoplasmic leucyl-tRNA synthetase. Purification and comparison of its catalytic, structural, and immunological properties with those of the chloroplastic enzyme.

The cytoplasmic leucyl-tRNA synthetase was purified from bean (Phaseolus vulgaris) leaves. After ammonium sulfate fractionation and chromatography on Sephadex G-50, DEAE-cellulose, hydroxylapatite, and phosphocellulose, complete purification was achieved by blue Sepharose CL-6B chromatography using specific elution with pure yeast tRNALeu1. The enzyme was purified 1050-fold and had a specific activity of 940 nmol of leucyl-tRNA formed/min/mg of protein. Polyacrylamide gel electrophoresis of the native enzyme showed one band, but the denatured enzyme showed two bands. These two protein bands are structurally related. The smallest protein appears to be a cleavage product from the largest one, suggesting the presence of a sensitive cleavage site in the cytoplasmic leucyl-tRNA synthetase. The cytoplasmic enzyme is a monomer (Mr = 130,000), larger than its chloroplastic counterpart (Mr = 120,000). The two enzymes differ in their substrate (tRNA) specificity, tryptic peptide map, and amino acid composition. Antibodies were raised against the cytoplasmic enzyme and against the chloroplastic enzyme and no cross-immunological reaction was detected, showing that the two enzymes do not share any antigenic determinant. Taken together, these results suggest that P. vulgaris cytoplasmic and chloroplastic leucyl-tRNA synthetases are coded for by different genes.

Amino Acids↗
PubMed1982

Purification and properties of chloroplast leucyl-tRNA synthetase from a higher plant: Phaseolus vulgaris.

The chloroplast leucyl-tRNA synthetase from Phaseolus vulgaris was purified by ammonium sulfate precipitation and chromatography on DEAE-cellulose, hydroxylapatite, and phosphocellulose. Finally, the pure enzyme was obtained after affinity chromatography on Blue Sepharose CL-6B using specific elution with a pure Escherichia coli tRNALeu isoacceptor. The specific activity of chloroplast leucyl-tRNA synthetase (1550 units/mg) is the highest ever obtained for a higher plant aminoacyl-tRNA synthetase. The purified enzyme has an optimal pH of 9.0 and Km values are, respectively, 2.6 X 10(-6) M for unfractionated E. coli tRNA, 0.85 X 10(-6) M for E. coli tRNA5Leu, 1.8 X 10(-4) M for ATP, and 1.4 X 10(-5) M for L-leucine. Chloroplast leucyl-tRNA synthetase is a large monomer which has a Mr of 122,000 as determined by electrophoresis on polyacrylamide gel in the presence of sodium dodecyl sulfate and urea and by gel filtration. Determination of Stokes radius, diffusion coefficient, and frictional ratio suggests that the enzyme structure is rather compact. The amino acid composition shows a relatively large proportion of apolar residues. Specific antibodies were raised in rabbits against the pure chloroplast leucyl-tRNA synthetase.

Amino Acids↗
PubMed1982

Chloroplastic and cytoplasmic valyl- and leucyl-tRNA synthetases from Euglena gracilis. Comparative study of their structural properties.

Chloroplastic and cytoplasmic valyl- and leucyl-tRNA synthetases purified from Euglena gracilis show a monomeric structure. The molecular weights of the two valyl-tRNA synthetases are identical (126,000) while those of the leucyl-tRNA synthetases are different (100 000 for the chloroplastic and 116 000 for the cytoplasmic enzyme). The tryptic maps and the amino acid compositions reveal differences between the chloroplastic valyl- and leucyl-tRNA synthetases and their cytoplasmic homologues. These results suggest that a chloroplastic aminoacyl-tRNA synthetase and its cytoplasmic counterpart are coded for by distinct genes.

Amino Acids↗
PubMed1981

Chloroplast leucyl-tRNA synthetase from Euglena gracilis. Purification, kinetic analysis, and structural characterization.

Euglena gracilis chloroplast leucyl-tRNA synthetase was purified to homogeneity by a series of steps including ammonium sulfate precipitation and chromatography on hydroxylapatite, DEAE-cellulose, Sepharose 6B, phosphocellulose, and Blue Dextran-Sepharose. The purified enzyme exhibits a specific activity of 1233 units/mg of protein, which is one of the highest specific activities obtained for an aminoacyl-tRNA synthetase prepared from plant cells. The enzyme has an apparent Km value of 8 x 10(-6) M for L-leucine, 1.3 x 10(-4) M for ATP, and 1.3 x 10(-6) M for tRNALeu. Chloroplast leucyl-tRNA synthetase appears to be a monomeric enzyme with a molecular weight of 100 000. The amino acid composition of chloroplast leucyl-tRNA synthetase has been determined. It is the first reported for a chloroplast aminoacyl-tRNA synthetase, and it reveals a relatively large proportion of apolar residues, as in the case of prokaryotic aminoacyl-tRNA synthetases.

Amino Acids↗
PubMed1981

Inactivation of beta-fucosidase by iodine, N-acetylimidazole and tetranitromethane. Evidence for the existence of essential tyrosine residues.

beta-Fucosidase (beta-D-fucoside fucohydrolase, EC 3.2.1.38) isolated from the digestive juice of Achatina balteata is markedly inactivated when the enzyme is preincubated with three different probes, specific for tyrosine residues in the experimental conditions used. An effective protection against inactivation of the enzyme is obtained in the presence of a substrate analogue. These data strongly suggest that beta-fucosidase possesses essential tyrosine residues.

Animals↗
PubMed1980

Simultaneous isolation of brush border and basolateral membrane from rabbit enterocytes. Presence of brush border hydrolases in the basolateral membrane of rabbit enterocytes.

By a slight modification of the procedure described by Gratecos et al. (Gratecos, D., Knibiehler, M., Benoit, V. and Sémériva, M. (1978) Biochim. Biophpys. Acta 512, 508-524), the basolateral and brush border membranes of rabbit enterocytes have been purified concomitantly from the same aliquot of mucosa. The two types of membrane have been obtained with the same yield (15%) and enrichment of specific markers (18-fold). The presence in the basolateral membrane of hydrolases known to be specific of the brush border membrane has been confirmed by using immunological techniques.

Animals↗
PubMed1980

Kinetic studies on beta-fucosidases of Achatina balteata.

beta-Fucosidases (beta-D-fucoside fucohydrolase, EC 3.2.1.38) isolated from the digestive juice of Achatina balteata catalyze hydrolysis of beta-D-fucosides, beta-D-glucosides and beta-D-galactosides but the values of kinetic parameters show that catalytic efficiency is maximum towards beta-D-fucosides. The results of mixed substrate incubation studied and inhibition by glycopyranoses indicate that there is at least one site at which all tested substrates are hydrolyzed. In the absence of inhibitor, the reciprocal plots exhibit a significant downward curvature. If a substrate analogue is present, the plots can be straight lines. These results are consistent with the presence on the enzyme molecule of at least two distinct sites for the substrate molecules, one being an active site and the other being either a second active site with different kinetic parameters or a modifier site. Also data are shown to fit quite well with the mechanism proposed for a mnemonic enzyme.

Animals↗
PubMed1978

Some physicochemical and structural properties of two beta-fucosidases from Achatina balteata.

Some properties of two beta-fucosidases from the digestive juice of a giant snail Achatina balteata were studied. Their amino acid composition was similar and both enzymes were shown to be rich in acidic and aromatic residues. Alanine was the single N-terminal residue in the two molecules. The enzymes appeared to be devoid of sialic acid but to contain galactose, glucose, mannose, fucose and hexosamines. The total sugar content was higher in beta-fucosidase I (21.6% w/w) than in beta-fucosidase II (12.7% w/w). Gel filtration and ultracentrifugation assays were consistent with an apparent molecular weight higher than 300 000 for beta-fucosidase I and of about 110 000 for beta-fucosidase II. On SDS polyacrylamide gel electrophoresis, beta-fucosidase I dissociated into two subunits (mol. wt. 190 000 and 170 000), whereas beta-fucosidase II showed a single band with a mol. w. of about 110 000. On the basis of these studies, it is concluded that the two forms are not structurally related proteins.

Amino Acids↗
PubMed1977

[Purification of two beta-D-glycosidases from the digestive juice of Achatina balteata].

Two beta-D-glycosidases have been purified from the digestive juice of Achatina balteata by acetone fractionation, ion exchange chromatography through DEAE-Sephadex A-50, ammonium sulphate fractionation and gel chromatography through Sephadex G-200. The preparations are homogeneous by p/lyacrylamide gel electrophoresis. Both enzymes are highly specific for the beta-D-anomeric configuration of the glycosidic linkage. They hydrolyse lactose, cellobiose and synthetic beta-D-galactosides, -glucosides and -fucosides at a pH optimum of 5,2 to 5,6 and are inactive on alpha-glycosides. The hydrolyzed substrates are recognized by the same catalytic site as shown by mutual competition studies between substrates and competitive inhibition observed with aldonolactones and glycopyranoses such as D-galactose, D-glucose and D-fucose. The different substrates are not hydrolyzed at the same rate by the two enzymes. They also differ by their electrophoretic mobility, their behaviour in gel chromatography and their stability towards pH and heat. The most salient property is the important beta-D-fucosidase activity of the two purified enzymes.

Animals↗
PubMed1975

[Characterization of some hydrolase activities in digestive juice of Achatina balteata].

The digestive juice of Achatina balteata, a giant snail of the West African Coast catalyses the hydrolysis of several natural and synthetic compounds. Enzymatic activities on lactose, o- and p-nitrophenyl-beta-D-galactoside, p-nitrophenyl-beta-D-glucoside, p-nitrophenyl-beta-D (and alpha-L-) fucoside, o-nitrophenyl-beta-D-xyloside, p-nitrophenyl-N-acetyl-beta-D-glucosaminide and phenolphthalein-glucuronide have been shown to be present. The effect of pH and substrate concentration on these activities were studied. The galactosidase, glucosidase and fucosidase activities were studied with respect to temperature, heat inactivation, pH stability and incubation with trypsin. Kinetic experiments suggest the presence of several galactosidase activities. This hypothesis is confirmed by specific staining after polyacrylamide gel electrophoresis. These activities showed a broad specificity towards galactosides and glucosides. The digestive juice showed no action on acetyl-L-tyrosine and benzoyl-L-arginine ethyl esters. However a small protease activity was observed on hemoglobine. No lipase activity was found. Sulfatase content was low compared to that of Helix pomatia.

Animals↗
PubMedDate not supplied

Somatostatin binding to dissociated cells from rat cerebral cortex.

A method has been developed for the study of somatostatin (SS) binding to dissociated cells from rat cerebral cortex. Binding of [125I] [Tyr11]SS to cells obtained by mechanical dissociation of rat cerebral cortex was dependent on time and temperature, saturable, reversible and highly specific. Under conditions of equilibrium, i.e., 60 min at 25 degrees C, native SS inhibited tracer binding in a dose-dependent manner. The Scatchard analysis of binding data was linear and yielded a dissociation constant of 0.60 +/- 0.08 nM with a maximal binding capacity of 160 +/- 16 fmol/mg protein. The binding of [125I] [Tyr11]SS was specific as shown in experiments on tracer displacement by the native peptides, SS analogues, and unrelated peptides.

Animals↗
PubMedDate not supplied

Butyrylcholinesterase from intestinal epithelial cells of quail, chick and duck: a comparative study during development.

1. Epithelial cells of avian intestine express non-specific cholinesterase (butyrylcholinesterase, BuChE) activity both in the embryo and adult animal. 2. Quail, duck and chick exhibit increased BuChE activity during the late embryonic period followed by decreased activity. The minimum value is reached after hatching at day 1 in quail, day 4 in chick and day 6 in duck. 3. The three species display marked sex-related differences mainly during the peri-hatching period. BuChE activity is higher in females than in males. 4. The three globular forms, G1, G2 and G4, are detected during the last days of embryonic development. After hatching, changes in the relative amounts of these forms are related to the disappearance of G2 and the gradual decrease of G4. In adult quail and duck, G4 is not detected and BuChE activity corresponds only to the G1 form. 5. The changes in BuChE activity and distribution of the molecular forms occur in a similar manner in all three species although the embryonic periods differ notably, suggesting that hormonal factors secreted during this period and involved in the preparation of hatching may regulate BuChE activity.

Animals↗
PubMedDate not supplied

Direct vascular actions of methyclothiazide and indapamide in aorta of spontaneously hypertensive rats.

In vitro experiments were designed to assess the inhibitory effect of the thiazide diuretics methyclothiazide (MCTZ), the hydrochlorothiazide (HCTZ), and the thiazide-related diuretic indapamide (IND) on contractile responses to norepinephrine (NE) and arginine vasopressin (AVP) of aortic rings from spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto rats (WKY). Changes in the tension of aortic ring preparations were measured isometrically. MCTZ (10(-4) M) induced endothelium-dependent inhibition of the vasoconstrictor responses to NE and AVP only in aortas from SHR, and the maximal vasoconstrictive effect of NE and AVP was decreased by 59 +/- 11% and 32.3 +/- 13%, respectively. Indapamide (10(-4) M) also induced endothelium-dependent inhibition of the contractile response to AVP in aortic rings from SHR, and the maximal vasoconstrictive effect of AVP was decreased by 33 +/- 5%. In contrast, HCTZ did not inhibit the contractile response to either NE or AVP, even at the highest concentration. This study provides evidence that methyclothiazide and indapamide inhibit the contractile response induced by norepinephrine and/or arginine vasopressin on SHR aortic preparations via an endothelium-dependent mechanism.

Animals↗