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Biomedical subjects

A Reglero

Publications and source records attributed to A Reglero.

At least 55 records · Page 3Linked to original sources
PubMed1989

In vitro synthesis of colominic acid by membrane-bound sialyltransferase of Escherichia coli K-235. Kinetic properties of this enzyme and inhibition by CMP and other cytidine nucleotides.

The membrane-bound sialyltransferase obtained from Escherichia coli K-235 grown in a chemically defined medium (ideal for colominic acid production) was studied. The in vivo half-life calculated for this enzyme was 20 h. Kinetic tests revealed (at 33 degrees C and pH 8.3) hyperbolic behaviour with respect to CMP-Neu5Ac (Km250 microM) and a transition temperature at 31.3 degrees C. The enzyme was inhibited by NH4+, some divalent cations and by several agents that react with thiol groups. Detergents and fatty acids also inhibited the sialyltransferase activity. In vitro synthesis of colominic acid is strongly inhibited by CMP by blocking the incorporation of [14C]Neu5Ac into a protein-complex intermediate and therefore into free polymer. CDP and CTP also inhibited (91% and 84%) this enzyme activity whereas cytosine and cytidine had no effect. CMP inhibition corresponded to a competitive model the calculated Ki was 30 microM. Incubations of protein[14C]Neu5Ac with CMP, CDP and CTP led to de novo synthesis of CMP-[14C]Neu5Ac. The presence of colominic acid, which usually displaces the reaction equilibrium towards polymer synthesis, did not affect this de novo CMP-[14C]Neu5Ac formation. CMP also inhibited in vivo colominic acid biosynthesis.

Cell Membrane↗
PubMed1989

Uptake of phenylacetic acid by Penicillium chrysogenum Wis 54-1255: a critical regulatory point in benzylpenicillin biosynthesis.

The transport system of phenylacetic acid (PA) in Penicillium chrysogenum was studied. Kinetic measurements were carried out "in vivo" at 25 degrees C in 0.06 M phosphate buffer at pH 6.5. Uptake was a linear function of time over 3 minutes and the Km was 5.2 microM. PA uptake was inhibited by 2,4-dinitrophenol, 4-nitrophenol, sodium azide, potassium cyanide. N-ethylmaleimide, amino acids, xylose and fatty acids whereas lactose and ribose stimulated it. Benzylpenicillin, phenoxymethylpenicillin, penicillins DF, K and 6-aminopenicillanic acid did not modify uptake whereas phenoxyacetic acid and many phenyl derivatives strongly inhibited the incorporation of PA. PA transport is an inducible system that is strictly regulated by the carbon source used for P. chrysogenum growth. Uptake is not induced by phenoxyacetic acid and is repressed by L-lysine. The absence of the PA transport system when P. chrysogenum is grown in the presence of readily metabolized sugars and its repression by L-lysine suggests that this is a critical regulatory point in the control of benzylpenicillin biosynthesis.

Amino Acids↗
PubMed1989

Phenylacetic acid transport system in Penicillium chrysogenum Wis 54-1255: molecular specificity of its induction.

The phenylacetic acid (PA) transport system of Penicillium chrysogenum is induced by PA, 2-hydroxyphenylacetic and 4-phenylbutyric acids but not by benzoic, phenoxyacetic acid and phenylpropionic acids. Substitution in the aromatic moiety (3-hydroxyphenylacetic, 4-hydroxyphenylacetic acids), replacement of the aromatic moiety by other rings (thiophene-2-acetic acid, indole-3-acetic or indole-3-butyric acids) or the presence of an amino group in the alpha-position (2-aminophenylacetic acid) eliminates inducing activity. 2-Phenylbutyric acid dose not induce the PA transport system indicating that fatty acid-beta-oxidation is needed to generate the authentic regulatory molecule (phenylacetyl-CoA) from 4-phenylbutyric acid. Furthermore, the uptake system synthesized in presence of PA, 2-hydroxyphenylacetic or 4-phenylbutyric acids is under carbon catabolic repression control and is also repressed by L-lysine suggesting that the three molecules induce in P. chrysogenum a single mechanism of transport.

Benzoates↗
PubMed1989

Repression of phenylacetic acid transport system in Penicillium chrysogenum Wis 54-1255 by free amino acids and ammonium salts.

The phenylacetic acid (PA) transport system in Penicillium chrysogenum is an inducible-system (see Fernández-Cañón et al.; preceding papers) which is repressed by free amino acids when these molecules are added to the complex fermentation broths at the induction time. L-Tyrosine, L-alpha-aminoadipic acid, L-tryptophan, L-phenylalanine and L-methionine are the molecules that cause the greatest delay in induction. The addition of Krebs-cycle intermediates to the complex fermentation broth did not affect the rate of induction with the exception of oxalacetic acid and citric acid which strongly increased it. Ammonium salts and acetate also repressed the biosynthesis of the enzymes involved in the PA uptake.

Amino Acids↗
PubMed1989

IV. Acyl-CoA: 6-APA acyltransferase of Penicillium chrysogenum: studies on substrate specificity using phenylacetyl-CoA variants.

Two different penicillins (p- and m-methylbenzylpenicillin) were obtained "in vitro" by direct enzymatic synthesis, using homogeneously pure acyl-CoA: 6-aminopenicillanic acid (6-APA) acyltransferase from Penicillium chrysogenum, 6-APA and p- or m-tolylacetyl-CoA. The Km for these substrates were 6 and 15 mM, respectively, indicating that the affinity of the enzyme for these two molecules is much lower that shown by phenylacetyl-CoA (0.55 mM). Furthermore, acyltransferase does not recognize o-tolylacetyl-CoA as a substrate suggesting that the position of the methyl group on the aromatic moiety may have a very important role in the formation of the enzyme-substrate complex.

Acetyl Coenzyme A↗
PubMed1988

A protein-sialyl polymer complex involved in colominic acid biosynthesis. Effect of tunicamycin.

A protein-NeuAc complex involved in colominic acid biosynthesis has been identified in membrane preparations of Escherichia coli K-235. This compound had an Mr (estimated by SDS/polyacrylamide-gel electrophoresis and autoradiography) of about 100,000 and played the role of an 'initiator' or 'primer' (endogenous acceptor) in the synthesis of the whole polymer. Incubations of E. coli membranes with CMP-[14C]NeuAc (CMP-N-[14C]acetylneuraminic acid) pointed to the existence of a protein fraction (primer acceptor) that linked residues of sialic acid (N-acetylneuraminic acid, NeuAc) up to a maximal size, later releasing them as low-Mr sialyl polymers (LMrS, Mr less than 10,000). In the presence of colominic acid (final acceptor) the radioactivity linked to the protein quickly decreased, appearing stoichiometrically bound to the whole polysaccharide. When membrane preparations were previously digested with Streptomyces proteinase or de-activated by heating (80 degrees C, 10 min), no incorporation of labelled NeuAc into trichloroacetic acid-insoluble material was detected. These results suggested that colominic acid molecules are synthesized while they are bound to a proteinaceous acceptor that is subsequently excised in the presence of colominic acid, generating the native protein. The antibiotic tunicamycin inhibited the biosynthesis of colominic acid, affecting the synthesis of this protein-(NeuAc)n intermediate. All these results are described here for the first time.

Bacterial Proteins↗
PubMed1988

Enzymatic synthesis of penicillins.

Different penicillins (phenylacetyl, 2-hydroxyphenylacetyl, 4-hydroxyphenylacetyl, phenoxyacetyl and 2-thiopheneacetylpenicillin) have been synthesized "in vitro" by direct N-acylation of 6-aminopenicillanic acid (6-APA) with the acyl group of several acyl-CoA derivatives. The enzyme that catalyzes these reactions, acyl-CoA: 6-APA acyltransferase of Penicillium chrysogenum, was purified to homogeneity (374-fold) and its amino acid composition is given. This protein accepts as substrates several aliphatic acids and different aromatic acids with the only requirement that an acetyl-CoA moiety must be present in the substrate molecule. Shortening or lengthening of the acyl moiety prevents the 6-APA-N-acylation reaction. The presence of an amino group in the alpha-position of the acetyl group does not allow this molecule to be used as substrate. However, different substitutions in the phenyl group (hydroxylation of the carbons 2 and 4) or its replacement by another aromatic ring (thiophene) were accepted with varying reactions rates in the acylation reaction when a 176-fold purified acyltransferase was employed. The homogeneity pure enzyme accepts as substrate thiophene acetyl-CoA but it did not 2-hydroxyphenyl and 4-hydroxyphenylacetyl-CoA. The presence of an oxygen atom between the aromatic and the acetyl moieties did not affect the catalysis.

Acyltransferases↗
PubMed1987

Uptake of N-acetylneuraminic acid by Escherichia coli K-235. Biochemical characterization of the transport system.

Kinetic measurement of the uptake of N-acetyl[4,5,6,7,8,9-14C]neuraminic acid by Escherichia coli K-235 was carried out in vivo at 37 degrees C in 0.1 M-Tris/maleate buffer, pH 7.0. Under these conditions uptake was linear for at least 30 min and the Km calculated for sialic acid was 30 microM. The transport system was osmotic-shock-sensitive and was strongly inhibited by uncouplers of oxidative phosphorylation [2,4-dinitrophenol (100%); NaN3 (66%]) and by the metabolic inhibitors KCN (84%) and sodium arsenate (76%). The thiol-containing compounds mercaptoethanol, glutathione, cysteine, dithiothreitol and cysteine had no significant effect on the sialic acid-transport rate, whereas the thiol-modifying reagents N-ethylmaleimide, iodoacetate and p-chloromercuribenzoate almost completely blocked (greater than 94%) the uptake of this N-acetyl-sugar. N-Acetylglucosamine inhibited non-competitively the transport of N-acetylneuraminic acid, whereas other carbohydrates (hexoses, pentoses, hexitols, hexuronic acids, disaccharides, trisaccharides) and N-acetyl-sugars or amino acid derivatives (N-acetylmannosamine, N-acetylcysteine, N-acetylproline and N-acetylglutamic acid) did not have any effect. Surprisingly, L-methionine and its non-sulphur analogue L-norleucine partially blocked the transport of this sugar (50%), whereas D-methionine, D-norleucine, several L-methionine derivatives (L-methionine methyl ester, L-methionine ethyl ester, L-methionine sulphoxide) and other amino acids did not affect sialic acid uptake. The N-acetylneuraminic acid-transport system is induced by sialic acid and is strictly regulated by the carbon source used for E. coli growth, arabinose, lactose, glucose, fructose and glucosamine being the carbohydrates that cause the greatest repressions in this system. Addition of cyclic AMP to the culture broth reversed the glucose effect, indicating that the N-acetylneuraminic acid-uptake system is under catabolic regulation. Protein synthesis is not needed for sialic acid transport.

Antimetabolites↗
PubMed1985

Carbohydrate contents, and glycosidase and glycosyl transferase activities in tissues from streptozotocin diabetic mice.

The contents of hexoses and hexosamines in brain, liver, and kidney of streptozotocin diabetic mice are significantly increased in comparison to the controls. These differences for hexoses contents in the heart are not significant. N-acetyl-beta-D-glucosaminidase and beta-D-glucosidase activities in brain, liver and kidney of diabetic mice are significantly higher when compared to the controls. However, beta-D-galactosidase activity is significantly lower in brain, liver, spleen and kidney of the diabetic mice, in comparison to the controls and similar in heart. alpha-D-Mannosidase activity of diabetic mice is significantly increased in spleen and heart and significantly decreased in liver and kidney. alpha-L-Fucosidase of diabetic mice shows higher activities, with significant differences, in liver and spleen; however, in heart and kidney the activities are significantly lower. Brain sialyltransferase and galactosyltransferase activities are significantly increased in diabetic mice; but for heart and kidney these differences are not significant. The activity for brain and kidney fucosyltransferase is not significant and that for the other assayed organs is significantly higher in comparison to the controls.

Acetylglucosaminidase↗
PubMed1985

Isolation and characterization of gangliosides from pig lymphocytes.

Two major gangliosides from pig spleen lymphocytes, accounting for 57% of the total lipid-bound sialic acids, were isolated and purified to homogeneity by column chromatography on DEAE-Sephadex and silica gel. They were identified as GM3 (II3Neu5GcLacCer), and GD3 (II3(Neu5Gc)2LacCer), by thin-layer chromatography in comparison with standards and by analysis of the constituent sugars. The major fatty acids of these gangliosides were stearic acid and myristic acid, respectively. In addition to these gangliosides, GD2 and bands comigrating on thin-layer chromatography with authentic GM2, GM1, GD1a and GD1b were found. These compounds also occur in pig peripheral blood lymphocytes, where, however, GD3 represents about 70% of the total lipid-bound sialic acid.

Animals↗
PubMed1984

Sialidase assay by luminescence in the low picomole-range of sialic acid. Its application to the measurement of this activity in influenza virus.

A new procedure for a sialidase assay, by bioluminescence, has been developed. The substrate, N- acetylneuraminyllactose (sialyllactose), hydrolysed by the sialidase activity, releases lactose. This lactose is hydrolysed with beta-galactosidase. The released galactose is oxidized with galactose dehydrogenase and NAD. The NADH produced in the last step is measured by a luminescence system, coupling two enzymes, NAD(P)H dehydrogenase (FMN) and luciferase. This microassay, which is specific, rapid, simple and ultra-sensitive, is a measure for amounts as little as (at least) 5 pmol of N-acetylneuraminic acid (corresponding to 0.15 ng of the released sialic acid). It uses commercialized reagents (non-radioisotopic) and avoids interferences common in other procedures. This method has been used for measuring sialidase activity directly on intact virus, avoiding inconvenient modifications produced in the extraction of the enzyme. The specific activity of sialidase of influenza virus X31 (H3N2), determined by this procedure, is 0.65 U/mg of total virus protein.

Hydrolysis↗
PubMed1983

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.

Adolescent↗
PubMed1983

Hydrolytic enzyme activities, mainly from lysosomal localization, in sera from patients who ingested a toxic oil.

Some hydrolytic enzyme activities, mainly typical of lysosomal localization, have been determined in blood sera from patients who ingested a rapeseed oil (denatured with anilines and treated by a thermal process), and in healthy subjects. beta-N-Acetylglucosaminidase, beta-D-glucosidase, beta-D-glucuronidase, alpha-L-fucosidase and leucine aminopeptidase activities were significantly higher when compared with controls (p less than 0.001); higher activities but not significant (p less than 0.2) differences were found for alpha-D-mannosidase and alkaline phosphatase. In contrast, beta-D-galactosidase, alpha-D-galactosidase, acid phosphatase and lipase showed lower activities than controls. The significance of these results is discussed.

Adolescent↗
PubMed1983

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.

Chemical Phenomena↗
PubMed1982

N-acetyl beta-D-glucosaminidase and alpha-L-fucosidase activities in relation to glycosylated hemoglobin levels and to retinopathy in diabetes.

N-Acetyl beta-D-glucosaminidase and alpha-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 beta-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 (HbA1).

Acetylglucosaminidase↗
PubMed1982

Studies on neuraminidase from influenza virus A(H3N2) obtained by two procedures.

1. Neuraminidase was obtained by (A) bromelain solubilization or (B) by treatment with N-lauroylsarcosine. 2. 5-N-acetyl-2-O-(3-methoxyphenyl)-alpha-D-neuraminic acid, employed as substrate, avoids the interference produced by the thiobarbituric acid method, and is not interfered by the ampholytes. 3. Only about 20% of original enzyme activity was lost after electrofocusing. The sample from procedure A showed two peaks, corresponding to pIs 4.4 and 5.6. The sample from procedure B, having a higher activity, showed only one peak at pI 4.4. 4. Samples A and B showed different Km and hydrolysis rate with N-acetylneuraminyl-lactose and glycophorin A. It was not found significantly different with other substrates: alpha 1-acid glycoprotein, brain gangliosides, 5-N-acetyl-2-O-(3-methoxyphenyl)-alpha-D-neuraminic acid and 2'-(4-methyl umbelliferyl)-alpha-D-N-acetylneuraminic acid.

Amino Acids↗
PubMed1982

[Glycosidases of various mollusks: general properties, kinetic studies and action on natural substrates].

The beta-N-acetylhexosaminidase, beta-glucuronidase, alpha-galactosidase, beta-galactosidase and alpha-L-fucosidase activities, in six different species of molluscs, have been studied. The optimum pH was acid in all cases, in agreement with the lysosomal origin of these enzymes. They generally show several pI in their isoelectrofocusing profiles. Kinetic studies with enzymes having several activities in one protein, i.e. beta-N-acetylhexosaminidase and beta-galactosidase, have been carried out with mixed substrates in order to determine the occurrence of several active sites. The action of these enzymes on glycosidic rests containing natural substrates has been studied by enzymatic hydrolysis.

Animals↗