Bernard Colas

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.

Kinetic studies on β-fucosidases of Achatina balteata

β-Fucosidases (β-d-fucoside fucohydrolase, EC 3.2.1.38) isolated from the digestive juice of Achatina balteata catalyze hydrolysis of β-d-fucosides, β-d-glucosides and β-d-galactosides but the values of kinetic parameters show that catalytic efficiency is maximum towards β-d-fucosides. The results of mixed substrate incubation studies 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.

Kinetic study of a thermostable β-glycosidase of Thermus thermophilus. Effects of temperature and glucose on hydrolysis and transglycosylation reactions

A β-glycosidase of a thermophile, Thermus thermophilus, belonging to the glycoside hydrolase family 1, was cloned and overexpressed in Escherichia coli. The purified enzyme (Ttβgly) has a broad substrate specificity towards β-D-glucoside, β-D-galactoside and β-D-fucoside derivatives. The thermostability of Ttβgly was exploited to study its kinetic properties within the range 25–80[emsp4 ]°C. Whatever the temperature, except around 60[emsp4 ]°C, the enzyme displayed non-Michaelian kinetic behavior. Ttβgly was inhibited by high concentrations of substrate below 60[emsp4 ]°C and was activated by high concentrations of substrate above 60[emsp4 ]°C. The apparent kinetic parameters (kcat and Km) were calculated at different temperatures. Both kcat and Km increased with an increase in temperature, but up to 75[emsp4 ]°C the values of kcat increased much more rapidly than the values of Km. The observed kinetics might be due to a combination of factors including inhibition by excess substrate and stimulation due to transglycosylation reactions. Our results show that the substrate could act not only as a glycosyl donor but also as a glycosyl acceptor. In addition, when the glucose was added to reaction mixtures, inhibition or activation was observed depending on both substrate concentration and temperature. A reaction model is proposed to explain the kinetic behavior of Ttβgly. The scheme integrates the inhibition observed at high concentrations of substrate and the activation due to transglycosylation reactions implicating the existence of a transfer subsite.

Acetylcholinesterase and butyrylcholinesterase in the gut mucosal cells of various mammal species: Distribution along the intestine and molecular forms

1. A cholinesterase activity was shown to be present in the homogenates of the gut mucosal cells from seven mammal species examined. 2. The distribution of the cholinesterase activity in the mucosal cells along the intestine differs from one species to another. This distribution is not correlated with that of the aminopeptidase which is a specific marker of the enterocyte plasma membranes. 3. Except rabbit, all the other species contain a (G4) globular tetrameric form and either a (G1) monomeric form (pig, ox) or a (G2) dimeric form (mouse, rat, sheep). Both (G1) and (G2) forms are found with the (G4) form in the mucosal cells of kitten and cat. The solubility characteristics of these various forms were studied by sucrose gradient centrifugations in the presence and the absence of 1% Triton X-100. 4. The mucosal cells from the studied species essentially possess either acetylcholinesterase (rabbit, kitten, cat) or butyrylcholinesterase (ox, pig, sheep, rat, mouse). These findings indicate that both enzymes probably present identical physiological functions in this cell type.

Lithium chloride effect on phenylethyl-B-D-galactoside synthesis by Aspergillus oryzae B-D-galactosidase in the presence of high lactose concentration

SummaryPhenylethyl-β-D-galactoside (PEGal) synthesis by transglycosylation can be catalysed by Aspergillus oryzae β-galactosidase and was studied using high concentrations of glycosyl donor (lactose). The amount of PEGal formed reached a maximum value after 1Oh in the presence of 0.5M lactose. Beyond this time, PEGal was slowly hydrolysed. LiCl between 0.4M and 0.7M in the reaction medium prevented the progressive disappearance of the product.

Transglycosylation reactions by exoglycosidases from the termite Macrotermes subhyalinus.

The ability of four exoglycosidases (β-galactosidase, β-glucosidase, α-glucosidase and invertase) from the termite Macrotermes subhyalinus to catalyse tranglycosylation reactions was tested using lactose, cellobiose, maltose and sucrose as glycosyl donors and 2-phenylethanol as glycosyl acceptor. The experimental conditions were optimized in relation to the time course of the reaction, pH and concentrations of glycosyl donor and acceptor. Whereas the hydrolytic activity was largely predominant over the transferase activity with β-galactosidase and β-glucosidase, the transglycosylation activity represented 68% with α-glucosidase. In addition, as demonstrated by the transglycosylation product formed, the hydrolysis of sucrose was catalysed by α-glucosidase and not by invertase. On the basis of this work, α-glucosidase from M. subhyalinus appears to be a valuable tool for the preparation of neoglycoconjugates.

Characterization of a strictly specific acid β-galactosidase from Achatina achatina

An acid beta-galactosidase was isolated from the digestive juice of Achatina achatina and purified to homogeneity by anion exchange, gel-filtration and hydroxyapatite chromatographies. This enzyme is soluble, as are the cytosolic beta-galactosidases, functions at acid pH like the lysosomal enzymes but differs from the other soluble animal beta-galactosidases in that it is highly specific for the beta-D-galactosyl residue. In addition, it cleaves the beta1-4 linkage much faster than the beta1-3 and beta1-6 linkages. The enzyme is a monomeric glycoprotein with a molecular mass of 120-125 kDa and the carbohydrate moiety makes up approximately 6% (w/w) of the protein. The amino acid composition displays an important amount of acidic/amide and hydroxy amino acid residues and a low content of basic residues. The enzyme activity is markedly affected by the ionic strength of the medium and the rate-pH curve was shifted towards higher pH values in the presence of added salt. Acid beta-galactosidase is capable of catalysing transgalactosylation reactions. The yields of galactosylation of hydroxy amino acid derivatives, catalysed by the enzyme in the presence of lactose as the glycosyl donor, were higher than those reported previously with conventional sources of beta-galactosidases. In addition, the pH optimum is different for the hydrolysis (pH 3.2) and transgalactosylation (pH 5.0) reactions. On the basis of this work, the enzyme could be used as a tool in the structural analysis of D-galactose-containing oligosaccharide chains, as well as for the synthesis of glycoconjugates.

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 Bakers 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.

Immunological evidence for structural differences between Euglena gracilis chloroplastic valyl- and leucyl-tRNA synthetases and their cytoplasmic counterparts

In a plant cell, there are at least two aminoacyltRNA synthetases (EC 6.1.1. . . .) specific for the same amino acid, one in the cytoplasm and the other in the chloroplasts. There is indirect evidence that both enzymes are coded for by the nuclear genome in spite of the existence of a protein synthesis machinery inside the chloroplast (for a general review, see [ 11). The chloroplastic aminoacyl-tRNA synthetases are known to differ from their cytoplasmic counterparts in their substrate (tRNA) specificity and their chromatographic mobility [ 1 ] but so far little was known about their structural similarities or differences. 2.1. Enzymes Chloroplastic and cytoplasmic ValRS and LeuRS were purified from green cells of Euglena gracilis Z as described in [2-51. One enzyme unit is defined as the amount of enzyme which catalyzes the aminoacylation of 1 nmol of tRNAin 1 min at 30°C.

Synthesis of β-galactosyl-(hydroxy amino acid) derivatives using β-galactosidase activity ofAchatina achatina digestive juice

SummaryThe transglycosylation activity of β-galactosidase fromAchatina achatina digestive juice was tested for glycosylating protected hydroxy amino acids. Attractive yields of β-galactosyl-(Z-Ser-OMe) (35%) and β-galactosyl-(Z-Hyp-OMe) (28%) could be obtained using lactose as glycosyl donor and the corresponding amino acid methyl esters N-protected by a benzyloxycarbonyl group (Z) as glycosyl acceptors.