Pedro Calvo

Serum β-N-acetylglucosaminidase, β-d-glucosidase, α-d-glucosidase, β-d-fucosidase, α-l-fucosidase and β-d-galactosidase levels in acute viral hepatitis, pancreatitis, myocardial infarction and breast cancer

Abstract The specific activities of several glycosidases ( β - N -acetylglucosaminidase, β- d -glucosidase, α- d -glucosidase, β- d -fucosidase, α- l -fucosidase and β- d -galactosidase) were determined in human sera from a control group of 10 normal subjects and in four groups, each of 10 patients, with acute viral hepatitis, acute pancreatitis, acute myocardial infarction and breast cancer. The results show significantly higher activities in acute viral hepatitis for β - N -acetylglucosaminidase, β- d -glucosidase and α- d -glucosidase ( p p d -glucosidase ( p d -fucosidase and α- l -fucosidase ( p β - N -acetylglucosaminidase, β- d -glucosidase, α- d -glucosidase, β- d -fucosidase and β- d -galactosidase ( p p p p p β - N -acetylglucosaminidase ( p

Kinetic evidence for two active sites in β-d-fucosidase of Helicella ericetorum

Abstract 1. 1. The kinetics of β- d -fucosidase of the snail H. eriretorum have been studied. The enzyme shows β- d -fucosidase, β- d -glucosidase and β- d -galactosidase activities, all associated in a single peak in both DEAE-cellulose chromatography and isoelectric focusing (pI 4.35). having the same optimal pH (5.0). 2. 2. With the corresponding p -nitrophenyl glycosides as substrates, β- d -fucosidase activity shows the lowest K m , the highest V max and the best V max / K m value: close activity values were obtained for β- d -glucosidase, however. β- d -galactosidase activity is much lower in this enzyme. 3. 3. All the kinetic evidence suggests that this enzyme has two active sites: a fuco-gluco site and a galacto site. 4. 4. β- d -fucosidase and β- d -glucosidase activities have similar K m , V max , V max / K m and K i values; these values are very different from those of β- d -galactosidase activity. β- d -fucosides and β- d -glucosides completely compete for a common active site in mixed-substrate experiments, while /J-D-galactosides only partially compete with both glycosides. 5. 5. With (δ- d -gluconolactone, the enzyme shows a hyperbolic mixed-type inhibition, mainly competitive for β- d -fucosidase and β- d -glucosidase activities (with the same inhibition sub-type), and predominantly non-competitive for β- d -galactosidase activity (with different inhibition sub-type). With δ- d -gluconolactone more inhibition of β- d -fucosidase and β- d -glucosidase activities was found, and with γ- d -galactonolactone, more inhibition of β- d -galactosidase activity was detected. 6. 6. The enzyme is activated by some carbohydrates, probably in relation with a transglycosylation mechanism.

Neuraminidase from influenza virus A (H3N2): specificity towards several substrates and procedure of activity determination.

Neuraminidase (acylneuraminyl hydrolase, EC 3.2.1.18) from the influenza virus A/Hong Kong/68 (H3N2) was purified after treatment of the purified virus with sarcosyl (sodium laurylsarcosinate), centrifugation at 110 000 x g, and chromatography on DEAE-Sephadex and Sephadex G-200. It migrated as a single component during electrophoresis on polyacrylamide gel, and its molecular weight was estimated about 270 000. The enzyme was thermolabile, the activity being reduced to 60% in 10 min at 50 degrees C. The purified neuraminidase had an apparent Km value of 4.1 . 10(-3) M for 5-N-acetyl-2-O-(3-methoxyphenyl)-alpha-D-neuraminic acid and was able to release sialic acid with linkages alpha 2-3, alpha 2-6 and alpha 2-8 (with very different efficiency) from fetuin, gangliosides, colominic acid, and bovine and porcine submaxillary mucins. The enzymic activity was measured by several procedures: (A) spectrophotometric determination at 340 nm of the NADH produced in the reaction catalysed by beta-galactose dehydrogenase on beta-galactose + NAD+, this beta-galactose was the product released from lactose by beta-galactosidase and lactose was the product of the neuraminidase activity on N-acetylneuraminyl-lactose; (B) determination of the colored quinone yielded by the liberated methoxyphenol with 4-aminoantipyrine (Santer, U.V., Yee-Foon, J. and Glick, M.C. (1978) Biochim. Biophys. Acta 523, 435-442); (C) periodate-thiobarbiturate procedures (Warren, L. (1959) J. Biol. Chem 234, 1971-1975 or Aminoff, D. (1961) Biochem. J. 81, 384-391). Some peculiarities of these methods are discussed.

β-Fucosidase, β-glucosidase and β-galactosldase activities associated in bovine liver☆

Abstract 1. 1. β- d -Fucosidase, β- d -glucosidase and β- d -galactosidase activities from bovine liver are associated in a single peak in isoelectric focusing. The isoelectric point is 4.35 for all these activities, suggesting that they are catalyzed by the same enzyme. 2. 2. This enzyme shows the optimal pH in the range 4.5–6.5 for all the above mentioned activities. 3. 3. The Km and Vmax are 0.26 mM and 31 mU mg−1, 0.10 mM and 24 mU mg−1, and 0.30 mM and 20 mM mg−1 for the p-nitrophenyl-fucoside, -glucoside and -galactoside, respectively. The glucoside derivative is the best substrate, with a Vmax/Km value of 0.24 mlβmg−1βmin−1. 4. 4. The Lineweaver-Burk profiles are convex upward in most cases, suggesting a substrate-activation model, and the presence of more than one binding site in the enzyme. 5. 5. The Ki for all the activities were determined with p-fucose, glucose and galactose as inhibitors. d -Fucose is the strongest inhibitor. The inhibition is competitive in all cases.

Purification, characterization and kinetics of β-N-acetylhexosaminidases A and B from the slug Arion rufus L

Two beta-N-acetylhexosaminidases have been purified to homogeneity and characterized, from the digestive gland of the slug A. rufus L., showing very high specific activities. Hexosaminidase A (Hex A) was purified 1300-fold with a yield of 12%, and hexosaminidase B (Hex B) was purified 1400-fold with a yield of 20%. Purified Hex A or Hex B run as a single protein band in polyacrylamide gel disc electrophoresis, showing different mobilities. The purified preparations do not show any of the other glycosidase activities present in the crude extract. beta-N-acetylglucosaminidase (GlcNAc-ase) and beta-N-acetylgalactosaminidase (GalNAc-ase) activities are always associated in a single peak for each enzyme form, with constant activity ratio, in all the purification steps, since they are catalyzed by the same enzyme (Hex A or Hex B). The optimal pH for both forms are 4.5 for GlcNAc-ase and 4.0 for GalNAc-ase activity. Hex B shows thermal and pH-stability higher than Hex A. The isoelectric points are 4.5 and 5.5 for A and B forms, respectively. The molecular weight is 150 000 for Hex A and 320 000 for Hex B. The amino acid composition of purified Hex A and B presents some differences concerning particularly Cys, Thr, Ser, Glu and Ile. The ratios Vmax/Km show that GlcNAc-ase is the main activity of both enzyme forms. beta-N-acetylglucosides and beta-N-acetylgalactosides completely compete for a common active site in mixed-substrates experiments. The Ki values are always coincident for GlcNAc-ase and GalNAc-ase activities, using competitive inhibitors (the corresponding lactones). These results strongly suggest that both activities are catalyzed by the same active site in both Hex A and B. Inhibition of the enzyme activities was found with the corresponding lactones, N-acetyl hexosamines, mannose, mannosides, HgCl2 and lead acetate; activation, with ribose, and with some chlorides and sulphates of divalent cations.

Characterization and kinetics of β-d-gluco/fuco/galactosidase from sheep liver

Abstract 1. 1. This enzyme shows β- d -glucosidase, β- d -fucosidase and β- d -galactosidase activities, all associated in a single peak in Sephadex G-200, DEAE-cellulose, concanavalin A-Sepharose chromatographies, and in high resolution isoelectric focusing (pI 4.56), having the optimal pH in the range 4.5–5.5. 2. 2. The enzyme is very stable under different conditions: (i) at pH in the range 5.5–7.0; (ii) in successive freezing-thawing cycles; (iii) at 4°C; (iv) after exhaustive ultrasonic treatment. It is not stable beyond 40°C, and in the presence of urea, Triton X-100, SDS or mercaptoethanol. 3. 3. HgCl 2 , KCN, Tris, maltose and the lactones were inhibitors of the enzyme. 4. 4. With glucose, fucose and galactose the inhibition is competitive. In addition, a transglycosylation mechanism seems to occur. 5. 5. The kinetic studies suggest a substrate-activation model and the presence of two primary active sites: fuco-gluco and galacto .

β-D-galactosidase isoenzymes in different sheep organs

Abstract 1. 1. Two types of β- d -galactosidase activity are present in sheep organs. 2. 2. A β- d -galactosidase that binds to concanavalin A and has an acidic pH-optimum, characteristic of a lysosomal hydrolase, predominates in sheep brain, kidney and spleen. 3. 3. A different β- d -galactosidase that does not bind to concanavalin A is also present in these tissues and accounts for 95% of the β- d -galactosidase in sheep liver. 4. 4. β- d -Glucosidase, β- d -fucosidase, β- d -xylosidase and α- l -arabinosidase activities also do not bind to concanavalin A. 5. 5. All five activities that do not bind to concanavalin A show the same pH-dependence, thermal stability and inhibition by Cl− ions and 2,5-dihydroxymethyl-3,4-dihydroxypyrrolidine (DMDP), a specific β- d -glucosidase inhibitor. 6. 6. It is postulated that a broad specificity glycosidase which has greatest activity towards β- d -glucosides accounts for all these activities. 7. 7. As the true β- d -galactosidase is not inhibited by DMDP it will be possible to use this inhibitor in a differential assay for the two types of β- d -galactosidase in sheep with a deficiency of the lysosomal β- d -galactosidase.

Hydrolysis of natural and synthetic substrates by α-l-fucosidase, β-d-glucuronidase and β-n-acetylhexosaminidase purified from molluscs

1. 1. Several mollusc glycosidases have been studied for their activities towards natural substrates. α-l-Fucosidases from Chamelea gallina, Tapes rhomboideus and Mytilus edulis hydrolyze oligosaccharides (di, tri and pentasaccharides) with α1 → 2, α1 → 3 and α1 → 4 bonds, fucose-containing glycopeptides from bovine thyroglobulin and the porcine submandibular mucin (devoid of sialic acid); α-l-fucosidase from Littorina littorea hydrolyzes fucose-containing glycopeptides from bovine thyroglobulin. 2. 2. β-d-Glucuronidase from L. littorea hydrolyzes hyaluronic acid, chondroitin 4-sulfate and heparin with a very low activity; however, it is much more active on oligosaccharides (from the above-mentioned macromolecules) containing non-reducing terminal glucuronyl residues. 3. 3. β-N-Acetylhexosaminidase from Helicella ericetorum acts mainly with an endo-hydrolase activity on β1 → 4N-acetylhexosamine linkages of ovalbumin, ovomucoid, chitin, hyaluronic acid and chondroitin 4. 4-sulfate; it has also a secondary exo-hydrolase activity on these substrates.

Comparative studies on blood serum α-l-fucosidases from several mammalian species

Abstract 1. 1. Peripheral blood serum α- l -fucosidases have been studied from various mammalian species: Sus scropha var domestica L. (pig), Capra hircus L. (goat), Bos taurus L. (bull, races Morucha and Charolais), Equus caballus L. (horse) and Equus asinus L. (donkey). 2. 2. Fluorimetric and spectrophotometric procedures were used for determination of α- l -fucosidases. 3. 3. α- l -Fucosidases were more active towards fluorescent substrates than towards chromogenic substrates. 4. 4. pH optima values of the enzymes are: (A) 5.5 for sera from all above-mentioned species when fluorescent substrates were employed; (B) 4.0 for goat, 4.5 for bull, 5.0 for pig and 4.5–5.0 for horse and donkey sera when chromogenic substrates were used. 5. 5. pH activity profiles are very similar for two races (Morucha and Charolais) of the same species ( Bos taurus L.) and also for two species of the same genus ( Equus caballus and Equus asinus L.) 6. 6. These serum α- l -fucosidases are very labile under heat treatment, even at 37°C.

Comparative studies on six blood serum glycosidases from several mammalian species

1. Peripheral blood serum alpha-D-galactosidase, beta-D-galactosidase, beta-D-glucosidase, alpha-D-mannosidase, beta-D-xylosidase and beta-D-glucuronidase have been studied with a comparative point of view from several mammalian species: Bos taurus L. (bull), Capra hircus L. (goat), Sus scropha var. domestica L. (pig) and man. 2. Fluorimetric and spectrophotometric procedures were used for determination of enzyme activities and pH optima. 3. Glycosidase activity was generally higher with fluorescent substrates than with chromogenic substrates. 4. alpha-D-mannosidase was the most active with both fluorescent and chromogenic substrates. 5. All the studied enzymes had the same pH optimum (4.0) when the chromogenic substrates were used. 6. pH optima of these glycosidases ranged from 3.0 to 5.5 when the fluorescent substrates were used.