Angel Reglero

Increased serum α-l-fucosidase and β-N-acetylglucosaminidase activities in diabetic, cirrhotic and gastric cancer patients

The activities of several glycosidases (α-l-fucosidase, α-D-mannosidase, α-D-galactosidase, β-d-gaIactosidase, β-N-acetylglucosaminidase and β-d-glucuronidase) were determined in human sera from 10 normal subjects and in three groups each of 10 patients with diabetes mellitus, hepatic cirrhosis and gastric carcinoma. The results show significantly higher activities in the patients for α-l-fucosidase (p < 0.001) and for β-N-acetylglucosammidase (p < 0.1, p < 0.001 and p < 0.05, respectively), and smaller or not significantly greater values for the other glycosidases.

Catabolism of D-glucose by Pseudomonas putida U occurs via extracellular transformation into D-gluconic acid and induction of a specific gluconate transport system

Pseudomonas putida U does not degrade D-glucose through the glycolytic pathway but requires (i) its oxidation to D-gluconic acid by a peripherally located constitutive glucose dehydrogenase (insensitive to osmotic shock), (ii) accumulation of D-gluconic acid in the extracellular medium, and (iii) the induction of a specific energy-dependent transport system responsible for the uptake of D-gluconic acid. This uptake system showed maximal rates of transport at 30 degrees C in 50 mM potassium phosphate buffer, pH 7.0. Under these conditions the K(m) calculated for D-gluconic acid was 6.7 microM. Furthermore, a different transport system, specific for the uptake of glucose, was also identified. It is active and shows maximal uptake rates at 35 degrees C in 50 mM potassium phosphate buffer, pH 6.0, with a K(m) value of 8.3 microM.

N-Acetyl β-d-glucosaminidase and α-l-fucosidase activities in relation to glycosylated hemoglobin levels and to retinopathy in diabetes

Abstract N -Acetyl β- d -glucosaminidase and α- l -fucosidase were determined in human sera from 25 control subjects, in 23 diabetic patients without retinopathy and in 22 diabetic patients with retinopathy. The results show significantly higher N -acetyl β- d -glucosaminidase activity in diabetic patients independently of the development of retinopathy and also independently of the length of diabetes. No correlation was found between either serum enzymes and serum glucose concentration and glycosylated hemoglobin (HbA,).

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.

Serum glycosidases in diabetes mellitus in relation to the retinopathy and to the length of the disease

The following glycosidase activities in sera have been studied: alpha-D-mannosidase, beta-D-glucuronidase, N-acetyl-beta-D-galactosaminidase, alpha-D-galactosidase, beta-D-galactosidase, alpha-D-glucosidase, beta-D-glucosidase and beta-D-fucosidase, in diabetic patients in relation to the presence of microangiopathy, evident by retinopathy, and to the length of the disease. A significant increase of all the enzyme activities, except for alpha-D-galactosidase was found. These elevations were independent of the development of retinopathy and the duration of the diabetic process.

A fluorometric procedure for measuring the neuraminidase activity: Its application to the determination of this activity in influenza and parainfluenza viruses

A fluorometric procedure for quantitating the amount of N-acetylneuraminic acid enzymatically released by the neuraminidase activity from N-acetylneuraminyl-lactose (sialyl-lactose) has been developed. The liberated lactose is hydrolyzed with beta-galactosidase, and the released galactose is oxidized with galactose dehydrogenase and NAD+; finally, the NADH produced is measured by fluorometry (excitation at 340 nm and analysis of emitted light at 465 nm). The fluorometric assay is about 10-fold more sensitive than the spectrophotometric procedure that measures NADH at 340 nm. It readily measures amounts as little as 2 nmol of sialic acid, and does not require the use of radioactive isotopes. Interferences due to sucrose or other substances, which cause errors in some cases with the use of the periodate-thiobarbiturate method for neuraminidase activity determination, are avoided. The procedure reported here provides a sensitive, rapid, and relatively simple method (feasible with commercialized reagents) for measuring the neuraminidase activity not only in purified samples from different sources but also directly in biological materials such as viruses. The technique has been tested with some viruses recently isolated belonging to Orthomyxoviridae or Paramyxoviridae families, known to be rich in neuraminidase. Reciprocally, this method can also be employed for determining the sialic acid concentration in acylneuraminyl-lactose-containing compounds when using purified neuraminidase for hydrolysis.

Fluorometric determination of phenylacetyl-CoA ligase from Pseudomonas putida: A very sensitive assay for a newly described enzyme

Phenylacetyl-CoA ligase (AMP-forming) from Pseudomonas putida is a newly described enzyme (Martinez-Blanco, H., Reglero, A., Rodriguez-Aparicio, L.B. and Luengo, J.M. (1990) J. Biol. Chem. 265, 7084-7090) specifically involved in the catabolism of phenylacetic acid. This enzyme catalyzes the formation of phenylacetyl-CoA in the presence of ATP, CoA, Mg2+ and phenylacetic acid. A rapid method of assaying this enzyme in partially purified preparations has been developed by coupling this reaction with adenylate kinase, pyruvate kinase and kinase and lactate dehydrogenase. The rate of phenylacetyl-CoA formation was measured indirectly by monitoring fluorometrically the NADH oxidation at 340 nm (excitation at 340 nm and analysis of the emitted light at 465 nm). The advantage of this method of assay over others (colorimetric, HPLC and spectrophotometric) is discussed.

Purification and some properties of α-l-fucosidase from Littorina littorea L.

Abstract 1. 1. The α- l -fucosidase from the marine mollusc Littorina littorea L. was purified approximately 225-fold; the purified enzyme was free of other glycosidase activities and showed a major band by polyacrylamide gel electrophoresis. 2. 2. Only one form of α- l -fucosidase has been detected by isoelectric focusing at pH 5.4 ± 0.1. 3. 3. The enzyme has an optimum pH of 6.5, Km of 3.0 × 10−4 M and Vmax of 2.6 μmol/mg per min with p-nitrophenyl α- l -fucopyranoside as substrate. 4. 4. l -fucose was a competitive inhibitor and Hg2+ was a no competitive inhibitor for α- l -fucosidase activity. 5. 5. This enzyme is stable in the range of pH values 4.0–9.0. Molecular weight of α- l -fucosidase by gel filtration was about 220,000. 6. 6. The carbohydrate composition of the enzyme was determined. The purified enzyme contains: glucose, 7.4%; galactose, 2.2%; mannose, 1.8%; and hexosamine, 2.2%.

Separation and properties of β-N-acetylglucosaminidases A, B and I from horse brain

Abstract 1. 1. Three forms of β-N-acetylglucosaminidase (A, B and I) were separated for the first time from horse brain by ion exchange chromatography on DEAE-cellulose. 2. 2. Form I has properties intermediate in the elution position from DEAE-cellulose, pI and thermal stability, under the assay conditions, between those of forms A and B. 3. 3. After isoelectric-focusing, specially forms A and I are transformed in more thermostable forms. 4. 4. d -Mannose, d -mannosamine, N- acetyl- d -glucosamine and α-methyl-d-mannoside have been found to inhibit the three above-mentioned forms. 5. 5. d -Glucosamine and d -galactosamine were weak inhibitors for β-N-acetylglucosaminidase B and I. 6. 6. Glucuronic acid was an inhibitor for forms A and I. 7. 7. The metabolic role of these inhibitions on specificities of β-N-acetylhexosaminidases towards natural substrates is discussed.

Inhibition of mammalian β-n-acetylglucos-aminidases A and B by mannose, α-methyl d-mannoside and mannosamine

Abstract 1. 1. d -Mannose. α-methyl d -mannoside and d -mannosamine have been found to inhibit β-N-acetylglucosaminidases A and B purified both from porcine placenta and bovine epididymis. 2. 2. The inhibition is competitive and the inhibitory constants are about 20 mM for mannose and α-methyl d -mannoside, and 2mM for mannosamine. 3. 3. The physiological role of this inhibitory effect is discussed.