José A. Cabezas

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.

Evaluation of patterns of urinary proteins by SDS-PAGE in rats of different ages

The patterns of urinary proteins in rats of different ages were examined on SDS gradient polyacrylamide gel electrophoresis coupled with silver staining. Proteins were fractionated into at least 26 bands. Densitometric measurements were used to characterize protein excretion patterns. The results showed that proteinuria in newborn, young and adult rats is predominantly tubular, consisting of low molecular-weight species. Conversely, late adults and old rats had a mixed glomerular pattern, with a steadily increasing excretion of albumin, IgG and transferrin, as was the case of other high molecular-weight proteins. Fragments of both immunoglobulins and albumin were found in all urine samples assayed. In 1 month old rats the percentage of Tamm-Hörsfall (T-H) protein was higher (P < 0.01) than in the remaining groups studied. In newborns, relatively high albumin, IgG and transferrin percentages were detected, as well as an alpha 1-acid glycoprotein and carbonic anhydrase excretion (P < 0.05 and P < 0.01 respectively) higher than that observed in the other age groups studied.

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.

Sero-epidemiological survey of influenza C virus infection in Spain

From an overall point of view, the epidemiological situation of influenza C virus infections in western Europe is hardly known. In some countries like Spain, no epidemiological survey has been carried out to determine whether influenza C virus does or does not circulate and cause infection in the considered geographical area. We thus decided to perform such a study. A total of 191 serum samples was collected from people (from 1.5 to 80 years old) living in Spain in October 1990. These sera were tested for the presence of antibodies to influenza C virus by hemagglutination-inhibition (HI) tests. Significant HI activity was found in 59.3 to 64.9% of the 191 tested sera and titres ranged from 20 to 320. The high prevalence of antibody as well as the highly significant titres indicate an intense circulation of influenza C virus in Spain. A significant difference was found between children/teenagers and adults.

Biochemical characterization of sheep platelet acetylcholinesterase after detergent solubilization

The biochemical characterization of detergent-solubilized acetylcholinesterase (AChE) from subcellular particles of sheep platelets and the effects of different effectors on AChE activity from solubilized platelet crude membranes have been undertaken and studied. Solubilization of AChE with detergent increased the thermal stability of the enzyme from all particulate fractions. Solubilized AChE from the mitochondria-granule fraction was the most thermostable at 55 degrees C. The Km values against acetylthiocholine chloride and the Arrhenius plot obtained were very similar for the AChE from all the solubilized fractions. There were no differences in the ability of solubilized AChE from different subcellular fractions to bind concanavalin A (Con A). In solubilized platelet crude membranes, benzyl alcohol was a potent AChE inhibitor at a concentration of 10(-2) M, whereas ethanol was not. Mg2+ cations and, to a lesser extent, Ca2+ and Mn2+ cations, activated AChE at concentrations higher than 1 mM. Serine hydrolase inhibitors and cholinesterase-specific inhibitors were very effective in the inactivation of AChE, whereas EDTA and EGTA had no effect. Of all the monosaccharides tested, only N-acetylneuraminic acid exerted an inhibitory effect on AChE activity. Immobilized-lectin binding studies demonstrated the interaction of solubilized crude membrane-bound AChE with Con A, lentil lectin and wheat germ agglutinin. Taken together, these data suggest the presence of a unique form of the membrane-bound AChE which has at least alpha-mannose and N-acetylglucosamine residues in the glycan chain.

Evaluation of the activities of eight lysosomal hydrolases in sera of humans, rats and pigs of different ages

The activities of 21 enzymes (belonging to four classes of enzymes) involved in different metabolic pathways were assayed in blood sera of healthy young and adult/elderly groups of humans, rats and pigs, to determine whether activity changes coinciding with changes in age and aging could be detected. In all three species analysed, measurable activities (performed by highly specific and sensitive techniques, generally spectrofluorimetric procedures) were found, usually following a decreasing order of: among glycosidases, beta-N-acetylglucosaminidase (NAG) > alpha-L-fucosidase > alpha-mannosidase > beta-glucuronidase > beta-galactosidase > alpha-galactosidase. In addition, among esterases very high values were found for arylesterase and acid phosphatase. By contrast, no measurable activity was found for the remaining enzymes assayed (8 hydrolases, 1 oxidoreductase, 3 transferases and 1 lyase). In the elderly group of humans, significantly higher activities (P < or = 0.05) were found for NAG, alpha-mannosidase and beta-glucuronidase in comparison to the adult and young groups. However, several activities in rats and all activities in pigs decreased with age. In conclusion, differences in the activities of 6 lysosomal glycosidases and 2 esterases (but no significant differences for another 13 enzymes belonging to several enzyme classes) are found in the sera of healthy humans, rats and pigs. These differences coincide with changes observed in aging.

Properties and kinetics of a neutral β-galactosidase from rabbit kidney

Summary A neutral β-galactosidase has been purified by concanavalin A-Sepharose affinity chromatography, DEAE-cellulose chromatography, Sephadex G-200 gel filtration and hydroxylapatite chromatography. The enzyme was purified 126-fold with a yield of about 21%. This form has a neutral optimal pH (7.5) and it is located in the cytosolic fraction. It shows a wide pH stability from pH 4.5 to 8.0, but it is very unstable at low pH values. Its isoelectric point is 4.9 and this value does not change on neuraminidase treatment. The estimated molecular weight was 47 000. The neutral form shows β- d -galactosidase, β- d -fucosidase and β- d -glucosidase activities, all of them associated in a single peak in all the purification steps. p -Nitrophenyl β- d -galactosides, p -nitrophenyl β- d -fucosides and p -nitrophenyl β- d -glucosides competed fully for a common active site in mixed-substrate experiments. Using γ- d -galactonolactone as competitive inhibitor the K i values were always coincident for the three activities. The effect of NaCl, methyl mannoside and some sugars (fucose, galactose and glucose) was studied.

Ganglioside and phospholipid composition of forebrain, cerebellum, and brain stem from adult and newborn rats.

The aim of this study was to elucidate whether sex or pregnancy state might affect the content and/or pattern of gangliosides from the forebrain, cerebellum and brain stem of rats. Adult male, mother (1-day after offspring) and nonpregnant rats of similar age were analyzed. Non-significant differences in ganglioside concentrations and patterns were found for the respective neural area of adult male and female rats except for a decrease in cerebellum and brain stem content from mothers and 12.0 months-old males, respectively. Thus, it seems that neither sex nor pregnancy hormones affect these parameters. By contrast, significant differences were found for pattern and ganglioside contents between adult (male and female) rats and newborns (1 day-old). Newborns showed a significant decrease in their forebrain (2.5-fold), cerebellum (2.0-fold) and brain stem (2.0-fold) ganglioside content when compared with adult (male and female) rats. Significant increases (p<0.001) were found in the phospholipid and cholesterol contents in the different brain areas in mothers versus their newborns. The phospholipid pattern also showed significant changes in all brain areas, with an increase (p<0.001) in phosphatidylethanolamine percentage in adult animals, among the main variations. An explanation for these facts is suggested.

Kinetic studies on the sialidase of three influenza B and three influenza A virus strains

Sialidase of influenza virus type A has been extensively studied through structural and kinetic approaches. However, sialidase of influenza virus type B has been less investigated. In this work, we have studied the activity and some properties (optimal pH, KM, Vmax, thermal stability) of sialidase in three influenza virus strains of type B (circulating in the period 1983–86) and also the activity and properties of sialidase from three virus strains of type A circulating at the same period of time.The results show that the activity and the Vmax was always higher for sialidase of type A viruses relative to those values of type B. Differences were also found for optimal pH and, in some cases, for thermal stability of the sialidase between strains belonging to the influenza viruses type A and B. However, the behaviour for the sialidase in all strains was very similar towards two competitive inhibitors. Thus, it could be suggested that the evolution pattern of the sialidase of both types of influenza viruses determines some modifications which result in a higher efficiency for sialidase of some strains of influenza virus type A, but maintaining in the two types of viruses a similar behaviour towards competitive inhibitors.

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 .