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E Villar

Publications and source records attributed to E Villar.

At least 55 records · Page 3Linked to original sources
PubMed1995

Irreversible thermal denaturation of Torpedo californica acetylcholinesterase.

Thermal denaturation of Torpedo californica acetylcholinesterase, a disulfide-linked homodimer with 537 amino acids in each subunit, was studied by differential scanning calorimetry. It displays a single calorimetric peak that is completely irreversible, the shape and temperature maximum depending on the scan rate. Thus, thermal denaturation of acetylcholinesterase is an irreversible process, under kinetic control, which is described well by the two-state kinetic scheme N-->D, with activation energy 131 +/- 8 kcal/mol. Analysis of the kinetics of denaturation in the thermal transition temperature range, by monitoring loss of enzymic activity, yields activation energy of 121 +/- 20 kcal/mol, similar to the value obtained by differential scanning calorimetry. Thermally denatured acetylcholinesterase displays spectroscopic characteristics typical of a molten globule state, similar to those of partially unfolded enzyme obtained by modification with thiol-specific reagents. Evidence is presented that the partially unfolded states produced by the two different treatments are thermodynamically favored relative to the native state.

Acetylcholinesterase↗
PubMed1995

Modulation of the neuraminidase activity of HN protein from Newcastle disease virus by substrate binding and conformational change: kinetic and thermal denaturation studies.

A statistical study of the enzyme kinetics of the soluble fraction of Haemagglutinin-Neuraminidase (HN) protein from Newcastle Disease virus reveals that a high substrate concentrations its neuraminidase activity follows substrate-inhibition kinetics, in which the binding of a second molecule of substrate to the protein inhibits its enzymatic activity. Results show that the enzymatic activity is modulated by the second substrate to a different extent when the protein is in different environments. Taken together with results obtained by thermal denaturation studies of HN under varying conditions the data show that conformational changes leading to a loss of rigidity of HN are concomitant with the loss of catalytic activity. Also, electrophoretic and sucrose gradient analyses show that the soluble domain of HN behaves as a monomer.

Kinetics↗
PubMed1994

Differential scanning calorimetric study of the thermal stability of xylanase from Streptomyces halstedii JM8.

The thermal stability of two xylanases with molecular masses of 45 (Xys1L) and 35 (Xys1S) kDa has been characterized thermodynamically by high-sensitivity scanning microcalorimetry in the pH range 3.0-9.0. Thermal denaturation of Xys1L reveals three thermodynamically independent domains, and that of Xys1S, which is a proteolytic fragment of Xys1L (without a C-terminal part), reveals two thermodynamically independent domains, each of which follows a two-state thermal unfolding process under our experimental conditions. Nevertheless, the thermodynamic parameters of unfolding for each domain do not fit some of the correlations obtained for most compact globular proteins. It is known that if delta Hres(T) and delta Sres(T) are plotted against temperature for a number of water-soluble compact globular proteins, they all have a common value at approximately 110 degrees C (383 K). Calculation of the variations in the enthalpy and entropy of unfolding per residue for each domain of xylanase with temperature gave us delta Hres(383) and delta Sres(383) values of approximately 3 kcal/(mol of residue) and 9 cal/(K.mol of residue), respectively. This is practically 2-fold larger than those apparent for most medium-sized globular protein values. These discrepancies might be related to features of the folded and/or unfolded states of the protein.

Calorimetry, Differential Scanning↗
PubMed1994

Domain structure and function of 10-formyltetrahydrofolate dehydrogenase.

10-Formyltetrahydrofolate dehydrogenase catalyzes the NADP(+)-dependent oxidation of 10-formyltetrahydrofolate to CO2 and tetrahydrofolate. Previous studies have shown that the enzyme binds the physiological pentaglutamate form of tetrahydrofolate product so tightly that it remains bound during size exclusion chromatography (Cook, R. J., and Wagner, C. (1982) Biochemistry 21, 4427-4434). In addition to the dehydrogenase activity, the enzyme from rat liver has been reported to exhibit both 10-formyltetrahydrofolate hydrolase and aldehyde dehydrogenase activities (Cook, R. J., Lloyd, R. S., and Wagner, C. (1991) J. Biol. Chem. 266, 4965-4973). We have purified the enzyme from rabbit liver and found that it catalyzes the same three reactions with similar kinetic constants and that it is a 99-kDa homotetramer, as reported previously for the rat and pig enzymes. Previous studies have suggested that the enzyme is composed of three domains and has separate folate binding sites for the dehydrogenase and hydrolase activities. We have investigated the domain structure of the rabbit enzyme. Differential scanning calorimetry reveals two thermal transitions, indicating the presence of two independently folded domains. The pentaglutamate form of tetrahydrofolate and NADP+ each stabilize one of the thermal transitions, showing that these ligands bind to separate domains. Limited proteolytic digestions by several proteases cleave the enzyme in a linker region between the two domains. After proteolytic cleavage, the domains no longer remain associated and do not catalyze the 10-formyltetrahydrofolate dehydrogenase reaction. Isolation and characterization of the intact domains revealed that the N-terminal domain only catalyzes the NADP(+)-independent 10-formyltetrahydrofolate hydrolase activity and the C-terminal domain only catalyzes the NADP(+)-dependent aldehyde dehydrogenase activity. The kinetic constants of these isolated domains are similar to those of the intact enzyme. Binding studies on the native enzyme using fluorescence and isothermal titration calorimetry indicated that the enzyme binds one molecule of tetrahydrofolate and two molecules of NADP+ per tetramer. Dissociation constants for both ligands were also determined by these methods.

Amino Acid Sequence↗
PubMed1994

Fusion between Newcastle disease virus and erythrocyte ghosts using octadecyl Rhodamine B fluorescence assay produces dequenching curves that fit the sum of two exponentials.

The kinetics of fusion between Newcastle disease virus and erythrocyte ghosts has been investigated with the octadecyl Rhodamine B chloride assay [Hoekstra, De Boer, Klappe, and Wilschut (1984) Biochemistry 23, 5675-5681], and the data from the dequenching curves were fitted by non-linear regression to currently used kinetic models. We used direct computer-assisted fitting of the dequenching curves to the mathematical equations. Discrimination between models was performed by statistical analysis of different fits. The experimental data fit the exponential model previously published [Nir, Klappe, and Hoekstra (1986) Biochemistry 25, 2155-2161] but we describe for the first time that the best fit was achieved for the sum of two exponential terms: A1[1-exp(-k1t)]+A2[1-exp(-k2t)]. The first exponential term represents a fast reaction and the second a slow dequenching reaction. These findings reveal the existence of two independent, but simultaneous, processes during the fusion assay. In order to challenge the model and to understand the meaning of both equation, fusion experiments were carried out under different conditions well known to affect viral fusion (changes in pH, temperature and ghost concentration, and the presence of disulphide-reducing agents or inhibitors of viral neuraminidase activity), and the same computer fitting scheme was followed. The first exponential equation represents the viral protein-dependent fusion process itself, because it is affected by the assay conditions. The second exponential equation accounts for a nonspecific reaction, because it is completely independent of the assay conditions and hence of the viral proteins. An interpretation of this second process is discussed in terms of probe transfer between vesicles.

Animals↗
PubMed1994

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.

Adolescent↗
PubMed1994

Domain structure of laccase I from the lignin-degrading basidiomycete PM1 revealed by differential scanning calorimetry.

The application of scanning calorimetry to investigate laccase I from the lignin-degrading basidomycete PM1 (CECT 2971) showed three thermal transitions beneath the overall endotherm following the previous heating of the sample up to 60 degrees C. The thermodynamic parameters of these three transitions satisfy a model of two-state independent unfolding, supporting a three-domain organization of the enzyme. It is shown that the catalytic site of laccase I is located in the domain with the thermally-induced transition at 76 degrees C.

Amino Acid Sequence↗
PubMed1994

Evaluation of a 22 kDa Dirofilaria immitis antigen for the immunodiagnosis of human pulmonary dirofilariosis.

Human pulmonary dirofilariosis usually appears as a solitary pulmonary nodule. A 22 kDa antigen from Dirofilaria immitis adult worms specifically recognized by three sera from individuals diagnosed with pulmonary dirofilariosis was identified by enzyme-linked-immunoelectrotransfer blot. This antigen was purified by elution from acrylamide gels and evaluated in enzyme-linked immunoassay using sera from 92 patients with pulmonary dirofilariosis and other pulmonary and parasitic diseases. Its use in the diagnosis of human pulmonary dirofilariosis should be considered.

Animals↗
PubMed1994

Stability of a low molecular mass cytotoxin from the sea anemone Radianthus macrodactylus and membrane-toxin interactions.

Differential scanning calorimetry, intrinsic fluorescence and SDS-polyacrylamide-gel-electrophoresis have been applied for the study of the low molecular mass cytotoxin from the sea anemone, Radianthus macrodactylus when in solution and in complexes with phospholipids and erythrocyte membranes. The thermodynamic parameters of toxin denaturation in solution have been obtained. It is shown that one molecule of this toxin withdraws 10 +/- 2 dipalmitoylphosphatidylcholine molecules through cooperative transition. The effect of toxin on dog erythrocytes causes a change in the polypeptide contents of erythrocyte ghosts.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1993

Natural infection of dogs by influenza C virus: a serological survey in Spain.

Two seroepidemiological surveys carried out so far, one in Japan, the other in France, gave a strong indication that dogs may be naturally infected by influenza C virus, considered to be exclusively human until recently. In this work, 101 serum samples were collected during winter 1989/1990 from dogs in Castilla y León, Spain. Sera were tested for the presence of antibodies to influenza C virus by Hemagglutination Inhibition (HI) test. Using antibody absorption by staphylococcal protein A, we demonstrated the specificity of the results. Significant HI activity was found in 56.3% of the 101 tested sera and titres ranged from 25 to 200.

Animals↗
PubMed1992

Increased influenza A virus sialidase activity with N-acetyl-9-O-acetylneuraminic acid-containing substrates resulting from influenza C virus O-acetylesterase action.

Influenza virus type C (Johannesburg/1/66) was used as a source for the enzyme O-acetylesterase (EC 3.1.1.53) with several natural sialoglycoconjugates as substrates. The resulting products were immediately employed as substrates using influenza virus type A [(Singapore/6/86) (H1N1) or Shanghai/11/87 (H3N2)] as a source for sialidase (neuraminidase, EC 3.2.1.18). A significant increase in the percentage of sialic acid released was found when the O-acetyl group was cleaved by O-acetylesterase activity from certain substrates (bovine submandibular gland mucin, rat serum glycoproteins, human saliva glycoproteins, mouse erythrocyte stroma, chick embryonic brain gangliosides and bovine brain gangliosides). A common feature of all these substrates is that they contain N-acetyl-9-O-acetylneuraminic acid residues. By contrast, no significant increase in the release of sialic acid was detected when certain other substrates could not be de-O-acetylated by the action of influenza C esterase, either because they lacked O-acetylsialic acid (human glycophorin A, alpha 1-acid glycoprotein from human serum, fetuin and porcine submandibular gland mucin) or because the 4-O-acetyl group was scarcely cleaved by the viral O-acetylesterase (equine submandibular gland mucin). The biological significance of these facts is discussed, relative to the infective capacity of influenza C virus.

Acetylesterase↗
PubMed1992

Increased serum N-acetyl-beta-D-glucosaminidase and alpha-D-mannosidase activities in obese subjects.

We have studied N-acetyl-beta-D-glucosaminidase and alpha-D-mannosidase activities in human sera from 35 control subjects, 47 normo- and hyperinsulinemic obese persons, and 12 diabetic patients after a fasting period of 12 h and at 30, 60, 90, and 120 min after an oral glucose overload. The results show a significantly higher activity of these 2 enzymes in obese subjects and diabetic patients, of similar magnitude, especially in those obese persons with a higher grade of obesity. Moreover, the activity of these glycosidases decreases in a similar way in all these 3 groups after the oral glucose overload.

Acetylglucosaminidase↗
PubMed1992

Age-related excretion of six glycosidases in rat urine.

The activity of beta-N-acetylglucosaminidase (NAG), beta-galactosidase, alpha-L-fucosidase, beta-glucuronidase, beta-glucosidase and alpha-mannosidase was determined in the urine of rats at progressive ages from newborn to old animals. The age-dependence of urinary creatinine, protein and pH values was also studied. Enzyme activity, related to urinary creatinine, was significantly higher in the newborn group than other ages. The excretion of NAG increased significantly in adult rats (3-6 months old) compared to young rats (1 month old). Most of the enzyme activities were diminished in old rats (25 months old). Increased proteinuria and creatinine excretion were observed in rats since 3 months of age. Age-related differences among enzyme activities therefore should be considered when these urinary glycosidases are to be studied in rats.

Acetylglucosaminidase↗
PubMed1992

Enzymatic glycosidase activities in experimental obesity.

Glycosidases are lysosomal enzymes that participate in the catabolism of glycoproteins and other glycoconjugates, and in some way may modify their activity in situations in which carbohydrate metabolism could be altered, such as the case of obesity. Using a fluorometric assay, a study was made of four glycosidase activities: N-acetyl-beta-D-hexosaminidase (NAG), alpha-mannosidase and alpha- and beta-glucosidase in the serum, pancreas, liver and kidney of 22 Zucker fa/fa genetically obese rats and of 23 fa/? controls, both with ages ranging between 13 and 15 weeks. After 12-14 hours fast and prior anaesthesia with sodium pentobarbital intraperitoneally, blood and the afore-mentioned organs were removed for enzymatic study of the serum and the organs after homogenization and centrifugation. In the serum a statistically significant increase in alpha-mannosidase (p < 0.0001) and alpha-glucosidase (p < 0.02) activities was found in the fa/fa obese rats as compared with the controls. No statistically significant differences were found in serum hexosaminidase activity between the two groups, and no serum beta-glucosidase enzymatic activity was detected. In liver, a decrease was observed in hexosaminidase (p < 0.002) and alpha-glucosidase (p < 0.01) activities in the obese rats as compared with the controls. In whole pancreas an increase was found in alpha-glucosidase activity in the obese rats with respect to the controls (p < 0.001), with no statistically significant differences in the hexosaminidase, alpha-mannosidase and beta-glucosidase activities.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗