Biomedical subjects

A Reglero

Publications and source records attributed to A Reglero.

At least 19 recordsLinked to original sources

PubMed1999

Determination of different amino sugar 2'-epimerase activities by coupling to N-acetylneuraminate synthesis.

A new procedure for quantitating the amount of N-acetyl-D-mannosamine (ManNAc) or ManNAc-6-phosphate produced by 2'-epimerase activities involved in sialic acid metabolism has been developed. The ManNAc generated by the action of N-acetyl-D-glucosamine (GlcNAc) and UDP-GlcNAc 2'-epimerases is condensed with pyruvate through the action of N-acetylneuraminate lyase and the sialic acid released is measured by the thiobarbituric acid assay. For the analysis of prokaryotic GlcNAc-6-phosphate 2'-epimerase, ManNAc-6-phosphate can also be evaluated by this coupled assay after dephosphorylation of the sugar phosphate. This system provides a sensitive, rapid, reproducible, specific and simple procedure (feasible with commercial reagents) for measuring amino sugar 2'-epimerases from eukaryotic and prokaryotic sources. The technique reported here permitted us to detect UDP-GlcNAc 2'-epimerase and GlcNAc 2'-epimerase in mammalian cell extracts and GlcNAc-6-phosphate 2'-epimerase in bacterial extracts.

Amino Sugars↗

PubMed1999

Uptake of N-acetyl-D-mannosamine: an essential intermediate in polysialic acid biosynthesis by Escherichia coli K92.

The N-acetyl-D-mannosamine (ManNAc) transport system of Escherichia coli K92 was studied when this bacterium was grown in a chemically defined medium containing ManNAc as carbon source. Kinetic measurements were carried out in vivo at 37 degrees C in 25 mM phosphate buffer, pH 7.5. Under these conditions, the uptake rate was linear for at least 15 min and the calculated Km for ManNAc was 280 microM. The transport system was strongly inhibited by sodium arsenate (97%), potassium cyanide (84%) and 2,4-dinitrophenol (88%) added at final concentrations of 1 mM (each). Analysis of bacterial ManNAc phosphotransferase activity revealed in vitro ManNAc phosphorylation activity only when phosphoenolpyruvate was present. These results strongly support the notion that ManNAc uptake depends on a specific phosphotransferase system. Study of specificities showed that N-acetylglucosamine and mannosamine specifically inhibited the transport of ManNAc in this bacterium. Analysis of expression revealed that the ManNAc transport system was induced by ManNAc, glucosamine, galactosamine, mannosamine and mannose but not by N-acetylglucosamine or N-acetylgalactosamine. Moreover, ManNAc permease was subject to glucose repression and cAMP stimulation. Full induction of the ManNAc transport system required the simultaneous presence of both cAMP and ManNAc.

Biological Transport↗

PubMed1999

Regulation of capsular polysialic acid biosynthesis by temperature in Pasteurella haemolytica A2.

The capsular polysaccharide of Pasteurella haemolytica A2 consists of a linear polymer of N-acetylneuraminic acid (Neu5Ac) with alpha(2-8) linkages. The production of this polymer is strictly regulated by the growth temperature and above 40 degrees C no production is detected. Analysis of the enzymatic activities directly involved in its biosynthesis reveals that Neu5Ac lyase, CMP-Neu5Ac synthetase and polysialyltransferase are involved in this regulation. Very low activities were found in P. haemolytica grown at 43 degrees C (at least 25 times lower than those observed when the growth temperature was 37 degrees C). The synthesis of these enzymes increased rapidly when bacteria grown at 43 degrees C were transferred to 37 degrees C and decreased dramatically when cells grown at 37 degrees C were transferred to 43 degrees C. These findings indicate that the cellular growth temperature regulates the synthesis of these enzymes and hence the concentration of the intermediates necessary for capsular polysaccharide genesis in P. haemolytica A2.

Bacterial Capsules↗

PubMed1998

Regulation of capsular polysialic acid biosynthesis by N-acetyl-D-mannosamine, an intermediate of sialic acid metabolism.

N-Acetyl-D-mannosamine (ManNAc) is a specific substrate for the synthesis of N-acetylneuraminic acid, the essential precursor of bacterial capsular polysialic acid (PA). When Escherichia coli K92 used ManNAc as a carbon source, we observed a dramatic reduction (up to 90%) in in vivo PA production. Experiments in which the carbon source was changed revealed that the maximal inhibitory effect occurred when this sugar was present in the medium before the logarithmic phase of bacterial growth had started. Enzymatic analysis revealed that high concentrations of ManNAc-6-phosphate inhibit NeuAc lyase, the enzyme that synthesizes NeuAc for PA biosynthesis in E. coli. These results indicate that ManNAc-6-phosphate is able to regulate NeuAc lyase activity and modulate the PA synthesis.

Bacterial Capsules↗

PubMed1998

Biochemical conditions for the production of polysialic acid by Pasteurella haemolytica A2.

The capsular polysaccharide of Pasteurella haemolytica A2 consists of a linear polymer of N-acetylneuraminic acid (Neu5Ac) with alpha(2-8) linkages. When the bacterium was grown at 37 degrees C for 90 h in 250 ml shake flasks at 200 rpm in Brain heart infusion broth (BHIB), it accumulated, attaining a level of 60 microg/ml. Release of this polymer was strictly regulated by the growth temperature, and above 40 degrees no production was detected. The pathway for the biosynthesis of this sialic acid capsular polymer was also examined in P. haemolytica A2 and was seen to involve the sequential presence of three enzymatic activities: Neu5Ac lyase activity, which synthesizes Neu5Ac by condensation of Nacetyl-D-mannosamine and pyruvate with apparent Km values of 91 mM and 73 mM, respectively; a CMP-Neu5Ac synthetase, which catalyzes the production of CMP-Neu5Ac from Neu5Ac and CTP with apparent Km values of 2 mM and 0.5 mM, respectively, and finally a membrane-associated polysialyltransferase, which catalyzes the incorporation of sialic acid from CMP-Neu5Ac into polymeric products with an apparent CMP-Neu5Ac Km of 250 microM.

Cell Division↗

PubMed1998

Identification, expression and tissue distribution of cytidine 5'-monophosphate N-acetylneuraminic acid synthetase activity in the rat.

We report the postnatal developmental profiles of N-acetylneuraminic acid cytidylyltransferase (EC 2.7.7.43) (CMP-Neu5Ac synthetase) in different rat tissues. This enzyme, which catalyses the activation of NeuAc to CMP-Neu5Ac, was detected in brain, kidney, heart, spleen, liver, stomach, intestine, lung, thymus, prostate and urinary bladder but not in skeletal muscle. Comparative analysis of the different specific activity profiles obtained shows that the expression of CMP Neu5Ac synthetase is tissue-dependent and does not seem to be embryologically determined. Changes in the level of sialylation during development were also found to be intimately related to variations in the expression of this enzyme, at least in brain, heart, kidney, stomach, intestine and lung.

Animals↗

PubMed1996

N-acetyl-D-neuraminic acid lyase generates the sialic acid for colominic acid biosynthesis in Escherichia coli K1.

Colominic acid is a capsular homopolymer from Escherichia coli K1 composed of alpha (2-8)-linked N-acetyl-D-neuraminic acid (NeuAc) residues. Recently, we have described that NeuAc synthesis in this bacterium occurs through the action of NeuAc lyase (EC 4.1.3.3) [ Rodríguez-Aparicio, Ferrero and Reglero (1995) Biochem. J.308, 501-505]. In the present work we analysed and characterized this enzyme. E. coli K1 NeuAc lyase is detected from the early logarithmic phase of growth, is induced by NeuAc and is not repressed by glucose. The enzyme was purified to apparent homogeneity (312-fold) using two types of hydrophobic chromatographies (butyl-agarose and phenyl-Sepharose CL-4B), gel filtration on Sephacryl S-200, and anion-exchange chromatography on DEAE-FPLC. The pure enzyme, whose amino acid composition and N-terminal amino acid sequence are also established, has a native molecular mass, estimated by gel filtration, of 135 +/- 3 kDa, whereas its molecular mass in SDS/PAGE was 33 +/- 1 kDa. The enzyme was able to synthesize and cleave NeuAc in a reversible reaction. The maximal rate of catalysis was achieved in 125 mM Tris/HCl buffer, pH 7.8, at 37 degrees C. Under these conditions, the K(m) values calculated for N-acetyl-D-mannosamine and pyruvate (condensation direction), and NeuAc (hydrolysis direction) were 7.7, 8.3 and 4.8 mM respectively. NeuAc synthesis by the pure enzyme was activated by Ca2+ and inhibited by Mn2+ and NeuAc, whereas the enzyme cleavage direction was inhibited by Ca2+, Mn2+ and pyruvate. The reaction products, NeuAc and pyruvate, and Ca2+ are able to regulate the direction of this enzyme (synthesis or cleavage of sialic acid) and, accordingly, to modulate colominic acid biosynthesis.

Amino Acid Sequence↗

PubMed1995

N-acetyl-D-neuraminic acid synthesis in Escherichia coli K1 occurs through condensation of N-acetyl-D-mannosamine and pyruvate.

Two enzymes have been found to be involved in bacterial N-acetyl-D-neuraminic acid (NeuAc) synthesis: NeuAc synthase, which condenses N-acetyl-L,D-mannosamine and phosphoenolpyruvate, and NeuAc lyase or NeuAc aldolase, which condenses N-acetyl-D-mannosamine and pyruvate. When we used Escherichia coli K1 crude extracts, we observed the generation of NeuAc in the presence of N-acetylmannosamine and both phosphoenolpyruvate (NeuAc synthase activity) or pyruvate (NeuAc lyase activity). However, when crude extracts were fractionated by Sephacryl S-200 chromatography, NeuAc synthase activity disappeared. A chromatographic peak of NeuAc synthase activity was detected when column fractions were re-tested in the presence of the active NeuAc lyase peak. Furthermore, crude extracts converted phosphoenolpyruvate into pyruvate. Pyruvate depletion, due to the addition of pyruvate decarboxylase to the NeuAc synthase reaction mixture, blocked NeuAc formation. Moreover, after NeuAc lyase immunoprecipitation no NeuAc synthase was detected. These findings suggest that NeuAc synthase is not present in E. coli K1 and therefore that NeuAc lyase is the only enzyme responsible for NeuAc synthesis in this bacterium.

Escherichia coli↗

PubMed1994

Catabolism of aromatics in Pseudomonas putida U. Formal evidence that phenylacetic acid and 4-hydroxyphenylacetic acid are catabolized by two unrelated pathways.

Phenylacetic acid (PhAcOH) and 4-hydroxyphenylacetic acid (4HOPhAcOH) are catabolized in Pseudomonas putida U through two different pathways. Mutation carried out with the transposon Tn5 has allowed the isolation of several mutants which, unlike the parental strain, are unable to grow in chemically defined medium containing either PhAcOH or 4HOPhAcOH as the sole carbon source. Analysis of these strains showed that the ten mutants unable to grow in PhAcOH medium grew well in the one containing 4HOPhAcOH, whereas four mutants handicapped in the degradation of 4HOPhAcOH were all able to utilize PhAcOH. These results show that the degradation of these two aromatic compounds in P. putida U is not carried out as formerly believed through a single linear and common pathway, but by two unrelated routes. Identification of the blocked point in the catabolic pathway and analysis of the intermediate accumulated, showed that the mutants unable to utilize 4HOPhAcOH corresponded to two different groups: those blocked in the gene encoding 4-hydroxyphenylacetic acid-3-hydroxylase; and those blocked in the gene encoding homoprotocatechuate-2,3-dioxygenase. Mutants unable to use PhAcOH as the sole carbon source have been also classified into two different groups: those which contain a functional PhAc-CoA ligase protein; and those lacking this enzyme activity.

Chromatography, High Pressure Liquid↗

PubMed1994

Inhibition of penicillin biosynthetic enzymes by halogen derivatives of phenylacetic acid.

The effect of phenylacetic acid (PAA) and several analogs on the activity of isopenicillin N synthase (IPNS) and acyl-CoA: 6-APA acyltransferase (AT) from Penicillium chrysogenum Wis 54-1255 has been tested. Whereas the substitution on the ring of a hydrogen atom by hydroxy-, methyl- or methoxy- groups did not cause any effect, the presence of halogens (Cl or Br) at positions 3 and/or 4 of PAA strongly inhibited these two enzymes. The replacement of hydrogen atoms by fluorine in certain positions also caused inhibition, but to a lesser extent.

Acyltransferases↗

PubMed1993

Characterisation of the gene encoding acetyl-CoA synthetase in Penicillium chrysogenum: conservation of intron position in plectomycetes.

Acetyl-coenzyme A synthetase (ACS; EC 6.2.1.1) from some plectomycete fungi is possibly involved in an accessory step of penicillin biosynthesis, in addition to its role in primary metabolism. We present the characterisation of the gene encoding this enzyme in Penicillium chrysogenum, which we designated acuA. Sequencing of genomic and cDNA clones showed that the coding region was interrupted by five introns, located at the same positions as those present in the Aspergillus nidulans homologue. This supports the possibility that the gene acquired its definitive mosaic organisation before the Penicillium/Aspergillus divergence. The mature transcript encodes a polypeptide with an M(r) of 74,287 which is 89.4% identical to its A. nidulans counterpart.

Acetate-CoA Ligase↗

PubMed1993

The action of Trypanosoma cruzi trans-sialidase on glycolipids and glycoproteins.

Addition of sialic acid residues in the human pathogen Trypanosoma cruzi glycoconjugates is mediated by a trans-sialidase and not by a CMP-sialic acid:glycoconjugate sialyltransferase. Incubation of trans-sialidase with N-[galactose-14C]acetyllactosamine and O-linked oligosaccharides, N-linked glycopeptides (both obtained from fetuin) or sialyllactose showed that the last three compounds were donors of sialic acid residues to the first one. Moreover, N- and O-linked oligosaccharides in asialofetuin and asialomucin, respectively, served as acceptors of sialic acid units. Gangliosides GM3, GD1a and GT1b but not GM2, GM1a nor GD1b donated sialic acid units to N-acetyllactos amine when incubated with trans-sialidase. This showed that only sialic acid units bound to terminal galactosyl residues were transferred. GM1a was converted to GD1a, and GD1b to GT1b when incubated with the appropriate donor. The fact that asialo-GM1a was converted to a ganglioside migrating as GD1a on thin-layer chromatography suggested that sialic acid units may be transferred to internal galactosyl residues, although once linked to those residues they can not be further transferred to other glycoconjugates. Sialic acid residues linked alpha 2,3- but not alpha 2,6- or alpha 2,8- were transferred by the trans-sialidase. Methyl beta-galactoside but not methyl alpha-galactoside served as acceptor of sialic acid units, thus suggesting that terminal alpha-linked galactosyl units in T. cruzi and mammalian glycoproteins are not sialylated by the enzyme. As the trans-sialidase employed in these experiments has been shown to be located on the external surface of the parasite and to be shed to the medium, the relatively broad specificity shown by the enzyme with respect to protein- and lipid-linked oligosaccharides strongly suggests that infection by T. cruzi might alter the sialic acid distribution in glycoproteins and glycolipids of the mammalian host.

Animals↗

PubMed1993

Polysialic acids.

1. Polysialic acids are linear homopolymers of N-acetylneuraminic acid (Neu5Ac), N-glycolylneuraminic acid (Neu5Gc) and deaminated neuraminic acid (KDN) residues joined by alpha 2,8, alpha 2-9 or alpha 2,8/alpha 2,9 ketosidic linkages. 2. They occur in glycoproteins of embryonic neural membranes (playing a role of neural cell adhesion molecules), in non-neural tissues (postnatal kidney), tumours, (neuroectodermal tumours), fish eggs and in the capsule of certain bacteria such as Neisseria meningitidis group B. 3. These polymers are synthesized through reactions which involve (a) the synthesis of sialic acid; (b) its activation to a cytidine monophosphate sugar nucleotide and (c) the polymerization of the different residues by a polysialyl-transferase complex. 4. Polysialic acids are involved in organogenesis and in cell growth. In several tissues they act as oncodevelopmental antigens, and in bacteria are also virulent determinants.

Animals↗

PubMed1993

Comparative analysis of the antibodies against capsular polysaccharides of Escherichia coli K-92 and K-235: an immunochemical method for the identification of polysialic acids.

1. The antibodies produced against the capsular poly-N-acetylneuraminic acid (poly-Neu5Ac) of E. coli K-92 (alpha 2-8-, alpha 2-9-linked) were 100-fold less sensitive than those obtained against E. coli K-235 capsular polysaccharide (CP) (alpha 2-8-linked) and recognized both kinds of polymers to a similar extent. 2. The partial hydrolysis of each purified polysaccharide revealed that E. coli K-92 CP is more labile at acidic pH than the polymer alpha 2-8-linked of E. coli K-235. 3. The antisera against CP from E. coli K-92 bound its own oligomers in which the number of Neu5Ac units was higher than three, whereas they only cross-reacted with the oligomers derived from E. coli K-235 containing a number of residues higher than 12. 4. The antisera against E. coli K-235 CP that recognized alpha 2-8 oligomers with a number of Neu5Ac residues higher than 5, also reacted, although very weakly, with those containing alpha 2-8 and alpha 2-9 linkages in which the carbon length was higher than (Neu5Ac)3. 5. Both types of antibodies were also able to recognize the native antigens in living bacteria and could be employed for the recognition of the type of linkage presents in different sialylpolymers.

Antibodies, Bacterial↗

PubMed1993

Purification of Pseudomonas putida acyl coenzyme A ligase active with a range of aliphatic and aromatic substrates.

Acyl coenzyme A (acyl-CoA) ligase (acyl-CoA synthetase [ACoAS]) from Pseudomonas putida U was purified to homogeneity (252-fold) after this bacterium was grown in a chemically defined medium containing octanoic acid as the sole carbon source. The enzyme, which has a mass of 67 kDa, showed maximal activity at 40 degrees C in 10 mM K2PO4H-NaPO4H2 buffer (pH 7.0) containing 20% (wt/vol) glycerol. Under these conditions, ACoAS showed hyperbolic behavior against acetate, CoA, and ATP; the Kms calculated for these substrates were 4.0, 0.7, and 5.2 mM, respectively. Acyl-CoA ligase recognizes several aliphatic molecules (acetic, propionic, butyric, valeric, hexanoic, heptanoic, and octanoic acids) as substrates, as well as some aromatic compounds (phenylacetic and phenoxyacetic acids). The broad substrate specificity of ACoAS from P. putida was confirmed by coupling it with acyl-CoA:6-aminopenicillanic acid acyltransferase from Penicillium chrysogenum to study the formation of several penicillins.

Caprylates↗

PubMed1992

Use of long-chain fatty acid-CoA ligase (AMP-forming) from Pseudomonas fragi for the 'in vitro' synthesis of natural penicillins.

Five different naturally occurring penicillins containing as side chains hexanoic, trans-3-hexenoic, heptanoic, octanoic or trans-3-octenoic acids have been synthesized 'in vitro' by coupling long-chain fatty acid-CoA ligase (AMP-forming) (EC 6.2.1.3) from Pseudomonas fragi (LFCoA-L) with acyl-CoA: 6-aminopenicillanic acid acyltransferase (AT) from Penicillium chrysogenum. The quantity of penicillin produced was directly related with the carbon length of the side chain precursor tested, being maximal with octanoic acid. Fatty acids with a lower length than C5 were not recognized as substrates and nor were certain aromatic molecules.

Acyl Coenzyme A↗

PubMed1992

Purification and characterization of the nuclear cytidine 5'-monophosphate N-acetylneuraminic acid synthetase from rat liver.

N-Acetylneuraminic acid cytidylyltransferase (EC 2.7.7.43) (CAMP-NeuAc synthetase) from rat liver catalyzes the formation of cytidine monophosphate N-acetylneuraminic acid from CTP and NeuAc. We have purified this enzyme to apparent homogeneity (241-fold) using gel filtration on Sephacryl S-200 and two types of affinity chromatographies (Reactive Brown-10 Agarose and Blue Sepharose CL-6B columns). The pure enzyme, whose amino acid composition and NH2-terminal amino acid sequence are also established, migrates as a single protein band on non-denaturing polyacrylamide gel electrophoresis. The molecular mass of the native enzyme, estimated by gel filtration, was 116 +/- 2 kDa whereas its Mr in sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 58 +/- 1 kDa. CMP-NeuAc synthetase requires Mg2+ for catalysis although this ion can be replaced by Mn2+, Ca2+, or Co2+. The optimal pH was 8.0 in the presence of 10 mM Mg2+ and 5 mM dithiothreitol. The apparent Km for CTP and NeuAc are 1.5 and 1.3 mM, respectively. The enzyme also converts N-glycolylneuraminic acid to its corresponding CMP-sialic acid (Km, 2.6 mM), whereas CMP-NeuAc, high CTP concentrations, and other nucleotides (CDP, CMP, ATP, UTP, GTP, and TTP) inhibited the enzyme to different extents.

Amino Acid Sequence↗

PubMed1992

Isolation and characterization of the acetyl-CoA synthetase from Penicillium chrysogenum. Involvement of this enzyme in the biosynthesis of penicillins.

Acetyl-CoA synthetase (ACS) of Penicillium chrysogenum was purified to homogeneity (745-fold) from fungal cultures grown in a chemically defined medium containing acetate as the main carbon source. The enzyme showed maximal rate of catalysis when incubated in 50 mM HCl-Tris buffer, pH 8.0, at 37 degrees C. Under these conditions, ACS showed hyperbolic behavior against acetate, CoA, and ATP; the Km values calculated for these substrates were 6.8, 0.18, and 17 mM, respectively. ACS recognized as substrates not only acetate but also several fatty acids ranging between C2 and C8 and some aromatic molecules (phenylacetic, 2-thiopheneacetic, and 3-thiopheneacetic acids). ATP can be replaced by ADP although, in this case, a lower activity was observed (37%). ACS in inhibited by some thiol reagents (5,5'-dithiobis(nitrobenzoic acid), N-ethylmaleimide, p-chloromercuribenzoate) and divalent cations (Zn2+, Cu2+, and Hg2+), whereas it was stimulated when the reaction mixtures contained 1 mM dithiothreitol, reduced glutathione, or 2-mercaptoethanol. The calculated molecular mass of ACS was 139 +/- 1 kDa, and the native enzyme is composed of two apparent identical subunits (70 kDa) in an alpha 2 oligomeric structure. ACS activity was regulated "in vivo" by carbon catabolite inactivation when glucose was taken up by cells in which the enzyme had been previously induced. This enzyme can be coupled "in vitro" to acyl-CoA:6-aminopenicillanic acid acyltransferase from P. chrysogenum, thus allowing the reconstitution of the functional enzymatic system which catalyzes the two latter reactions responsible for the biosynthesis of different penicillins. The ACS from Aspergillus nidulans can also be coupled to 6-aminopenicillanic acid acyltransferase to synthesize penicillins. These results strongly indicate that this enzyme can catalyze the activation (to their CoA thioesters) of some of the side-chain precursors required in these two fungi for the production of several penicillins. All these data are reported here for the first time.

Acetate-CoA Ligase↗