Biomedical subjects

A Reglero

Publications and source records attributed to A Reglero.

At least 37 records · Page 2Linked to original sources

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↗

PubMed1992

Characterization of an inducible transport system for glycerol in Streptomyces clavuligerus. Repression by L-serine.

Streptomyces clavuligerus NRRL 3585 grown in a chemically defined medium containing glycerol as the sole carbon source transported this molecule by two different systems. One of these was constitutive with a very low uptake efficiency and insufficient to attend to the metabolic requirements of this bacterium (constitutive glycerol transport system) and the other (glycerol transport system (GTS)) active and specifically induced by D-glycerol which is responsible for the transport of more than 90% of the glycerol taken up the cells. GTS was seen to have an optimal pH and temperature of 7.0 and 30 degrees C, respectively, and its Km was 14 microM. It was repressed by L-serine and addition of this amino acid to the culture broth (10 mM) inhibited the growth of S. clavuligerus but not that of other species of Streptomyces.

Biological Transport, Active↗

PubMed1991

H.p.l.c. of oligo(sialic acids). Application to the determination of the minimal chain length serving as exogenous acceptor in the enzymic synthesis of colominic acid.

A rapid, sensitive and easy h.p.l.c. method was developed for the quantitative analysis of oligosialic acids. This procedure which permits the complete separation (in 23 min) of several sialyloligomers with a degree of polymerization of between 1 and 16, has been employed to establish the minimal chain length of oligomer accepted, as an exogenous acceptor, by Escherichia coli K-235 sialytransferase complex (ST) leading to the synthesis in vitro of colominic acid. We showed that this membrane-bound enzyme catalyses the direct transfer of Neu5Ac residues (one by one) from CMP-Neu5Ac to an exogenous acceptor molecule which contains at least three Neu5Ac residues. Free Neu5Ac or (Neu5Ac)2 were not recognized as substrates, whereas the maximal rate of polymer elongation was achieved when (Neu5Ac)5 was used as substrate.

Chromatography, High Pressure Liquid↗

PubMed1991

Synthesis of 3-furylmethylpenicillin using an enzymatic procedure.

3-Furylmethylpenicillin was synthesized in vitro from 3-furylacetic acid, 6-aminopenicillanic acid (6-APA), CoA, ATP and Mg2+. The reaction was catalyzed in two steps by the enzymes phenyl-acetyl-CoA ligase (PCL) from Pseudomonas putida and acyl-CoA: 6-APA acyltransferase (AT) from Penicillium chrysogenum. PCL catalyzes the activation of 3-furylacetic acid to 3-furylacetyl-CoA (3-F-CoA) and AT acylates the amino group of 6-APA with the 3-furylacetyl moiety of 3-F-CoA, releasing CoA and 3-furylmethylpenicillin.

Acyltransferases↗

PubMed1991

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.

Acetyl Coenzyme A↗

PubMed1991

A study of thyroglobulin and peroxidase activity in the thyroid tissue of patients with non-endemic non-toxic nodular goitre.

1. A study was made of the activity of the enzyme thyroid peroxidase and of the concentration, carbohydrate composition and the degree of iodination of thyroglobulin in the thyroid glands of 60 patients with non-endemic non-toxic goitre in the nodular phase and in those of 25 control subjects. 2. Thyroid peroxidase activity was determined by the guaiacol assay and was significantly higher in patients with non-endemic non-toxic goitre than in control subjects (3.60 +/- 2.51 and 2.07 +/- 1.08 mumol of guaiacol oxidized min-1 g-1 of tissue, respectively; ranges 0.16-10.57 and 0.52-4.85 mumol of guaiacol oxidized min-1 g-1 of tissue, respectively; P less than 0.05). 3. Thyroglobulin was purified by precipitation with ammonium sulphate and Sephadex G-200 gel filtration. Two protein peaks were obtained which were identified as thyroglobulin and measured by radioimmunoassay. The concentration of thyroglobulin in the first peak was 98.94 (SD 84.87, range 0.60-455.54) mg/g of tissue for the patients with non-endemic, non-toxic goitre and 51.41 (SD 28.34, range 14.99-106.39) mg/g of tissue for the control subjects (P less than 0.01). The second peak showed 1.26 (SD 1.27, range 0.09-6.50) mg of thyroglobulin/mg of tissue for the group with non-endemic non-toxic goitre and 0.51 (SD 0.25, range 0.15-0.98 mg of thyroglobulin/mg of tissue) for the control subjects (P less than 0.01). 4. The carbohydrate composition of thyroglobulin was determined by acid hydrolysis and colorimetry, evaluating the levels of hexoses, hexosamines and sialic acid.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1991

In vitro enzymatic synthesis of new penicillins containing keto acids as side chains.

Seven different penicillins containing alpha-ketobutyric, beta-ketobutyric, gamma-ketovaleric, alpha-ketohexanoic, delta-ketohexanoic, epsilon-ketoheptanoic, and alpha-ketooctanoic acids as side chains have been synthesized in vitro by incubating the enzymes phenylacetyl coenzyme A (CoA) ligase from Pseudomonas putida and acyl-CoA:6-aminopenicillanic acid acyltransferase from Penicillium chrysogenum with CoA, ATP, Mg(2+), dithiothreitol, 6-aminopenicillanic acid, and the corresponding side chain precursor.

Keto Acids↗

PubMed1991

Acyl-CoA: 6-APA acyltransferase from Penicillium chrysogenum studies on its hydrolytic activity.

Acyl-CoA: 6-APA acyltransferase (AT) from Penicillium chrysogenum Wis 54-1255 catalyzes the hydrolysis of different acyl-CoA derivatives generating, in the absence of 6-APA, free acid and CoA. The hydrolytic efficiency of AT is highest for acyl-CoA variants in which the acyl-moiety is higher than six carbon atoms. The maximal rate of catalysis was achieved in 50 mM Tris-HCl buffer, pH 8.5 at 35 degrees C. Unlike the AT activity, the acylase activity has a different optimum temperature and substrate specificity and dithiothreitol is not required for the reaction.

Acyl Coenzyme A↗

PubMed1991

"In vitro" synthesis of different naturally-occurring, semisynthetic and synthetic penicillins using a new and effective enzymatic coupled system.

Forty-seven different penicillins, including some of great clinical importance, have been synthesized "in vitro" by coupling the newly described enzyme phenylacetyl-CoA ligase (PCL) from Pseudomonas putida and acyl-CoA: 6-aminopenicillanic acid (6-APA) acyltransferase (AT) from Penicillium chrysogenum. Incubations were carried out at 30 degrees C in 50 mM HCl-Tris buffer pH 8.0. The reaction mixtures contained 6-APA, CoA, ATP, dithiothreitol, Mg2+ and the corresponding penicillin side-chain precursor. This is the first description of the enzymatic synthesis of all the natural penicillins known, many of the semisynthetic until now reported, and some penicillins that could only be currently obtained by chemical synthesis. The efficiency of this prokaryotic-eukaryotic enzymatic-coupled system and its application to the synthesis of different beta-lactam antibiotics are discussed.

Acyltransferases↗

PubMed1990

Purification and biochemical characterization of phenylacetyl-CoA ligase from Pseudomonas putida. A specific enzyme for the catabolism of phenylacetic acid.

A new enzyme, phenylacetyl-CoA ligase (AMP-forming) (PA-CoA ligase, EC 6.2.1-) involved in the catabolism of phenylacetic acid (PAA) in Pseudomonas putida is described and characterized. PA-CoA ligase was specifically induced by PAA when P. putida was grown in a chemically defined medium in which phenylacetic acid was the sole carbon source. Hydroxyl, methyl-phenylacetyl derivatives, and other PAA close structural molecules did not induce the synthesis of this enzyme and neither did acetic, butyric, succinic, nor fatty acids (greater than C5 atoms carbon length). PA-CoA ligase requires ATP, CoA, PAA, and MgCl2 for its activity. The maximal rate of catalysis was achieved in 50 mM HCl/Tris buffer, pH 8.2, at 30 degrees C and under these conditions, the Km calculated for ATP, CoA, and PAA were 9.7, 1.0, and 16.5 mM, respectively. The enzyme is inhibited by some divalent cations (Cu2+, Zn2+, and Hg2+) and by the sulfhydryl reagents N-ethylmaleimide, 5,5'-dithiobis(2-nitrobenzoic acid), and p-chloromercuribenzoate. PA-CoA ligase was purified to homogeneity (513-fold). It runs as a single polypeptide in 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and has a molecular mass of 48 +/- 1 kDa. PA-CoA ligase does not use as substrate either 3-hydroxyphenylacetic, 4-hydroxyphenylacetic, or 3,4-dihydroxyphenylacetic acids and shows a substrate specificity different from other acyl-CoA-activating enzymes. The enzyme is detected in P. putida from the early logarithmic phase of growth and is repressed by glucose, suggesting that PA-CoA ligase is a specific enzyme involved in the utilization of PAA as energy source.

Amino Acids↗

PubMed1990

Carbon catabolite regulation of phenylacetyl-CoA ligase from Pseudomonas putida.

Phenylacetyl-CoA ligase (PA-CoA ligase) from P. putida U is a newly described enzyme involved in the aerobic catabolism of phenylacetic acid. The enzyme was specifically induced when P. putida was grown in a chemically defined medium containing phenylacetic acid as the sole carbon source. The induction of PA-CoA ligase was delayed by adding easily metabolizable carbon sources to the medium; the effect was more drastic in the presence of glucose. Glucose did not cause catabolic inactivation but rather catabolic repression, this effect being reversed by cAMP.

Aerobiosis↗

PubMed1990

Purification and properties of beta-N-acetylhexosaminidase A from pig brain.

1. Adult pig brain beta-N-acetylhexosaminidase was separated into four different forms by ion exchange chromatography on diethylaminoethyl-cellulose. 2. Form A was purified 1300-1500 fold by an unusual procedure, the technique of ampholyte displacement, followed by chromatography on concanavalin A Sepharose. 3. The enzyme catalyses the hydrolysis of both beta-N-acetylglucosaminides and beta-N-acetylgalactosaminides. 4. The kinetic studies support the evidence of the association of both activities to a single protein, and at the same active site. 5. A natural substrate, N,N'-diacetylchitobiose, is also hydrolyzed, but not ovalbumin. 6. This enzyme may be considered as an exoglycosidase.

Ampholyte Mixtures↗

PubMed1990

Design of an enzymatic hybrid system: a useful strategy for the biosynthesis of benzylpenicillin in vitro.

A hybrid (prokaryotic-eukaryotic) enzyme system leading to the production of benzylpenicillin has been developed. In vitro synthesis of penicillin G was achieved by incubating 6-aminopenicillanic acid, CoA, phenylacetic acid, homogeneously pure phenylacetyl-CoA ligase (PA-CoA ligase) from Pseudomonas putida and acyl-CoA:6-APA acyltransferase (AT) from Penicillium chrysogenum. Benzylpenicillin was also obtained when AT was coupled with PA-CoA ligase and isopenicillin N-synthetase (IPNS). This is the first description of an in vitro assay that, using enzymes of different microbial origin, mimics the three last enzymatic steps leading to the biosynthesis of penicillin G in P. chrysogenum.

Acyltransferases↗

PubMed1990

High production of polysialic acid [Neu5Ac alpha(2-8)-Neu5Ac alpha(2-9)]n by Escherichia coli K92 grown in a chemically defined medium. Regulation of temperature.

The capsular polysaccharide of Escherichia coli K92 consists of a linear polymer of Neu5Ac with alternating alpha(2-8) and alpha(2-9) linkages. It accumulates when the bacterium is grown at 37 degrees C in a defined medium containing D-xylose and L-asparagine as carbon and nitrogen sources. Release of the capsular polymer into the medium was maximal (450 micrograms x ml-1) in the stationary phase of growth (76 h). This medium could be useful for obtaining sufficient polymer to develop effective vaccines. The enzyme, CMP-Neu5Ac synthetase, was not detected in cells grown at 20 degrees C. The lack of this enzyme explains the absence of polymer biosynthesis when the bacterium was grown at 20 degrees C.

Antigens, Bacterial↗

PubMed1990

Aliphatic molecules (C-6 to C-8) containing double or triple bonds as potential penicillin side-chain precursors.

Three different hexenoyl-CoA derivatives (trans-2-hexenoyl-CoA, trans-3-hexenoyl-CoA and trans-trans-2,4-hexadienoyl-CoA), two octenoyl-CoA (trans-2-octenoyl-CoA, trans-3-octenoyl-CoA) and 2-octynoyl-CoA were tested as substrates of the enzyme acyl-CoA: 6-Aminopenicillanic acid acyltransferase (AT) from Penicillium chrysogenum. Only trans-3-hexenoyl-CoA and trans-3-octenoyl-CoA were recognized by AT and efficiently converted into penicillin F and octenoylpenicillin, respectively. The Km values for these substrates were 0.6 and 0.5 mM, suggesting that the affinity of AT for these molecules is similar to that reported for phenyl acetyl-CoA, octanoyl-CoA and hexanoyl-CoA (0.5, 0.6, and 1 mM, respectively). The absence of enzymatic activity shown by AT with the other acyl-CoA derivatives tested is due to the different position of the double or triple bond(s) in their aliphatic chains. The influence of the free rotation round the bond C-2-C-3 and possibility of planar conformation in such molecules and the importance in the formation of the enzyme-substrate complex is discussed.

Acyl Coenzyme A↗

PubMed1990

Biosynthesis of benzylpenicillin (G), phenoxymethylpenicillin (V) and octanoylpenicillin (K) from glutathione S-derivatives.

"In vitro" synthesis of benzylpenicillin and phenoxymethylpenicillin has been carried out by direct N-acylation of 6-aminopenicillanic acid (6-APA) with S-phenylacetyl- and (S-phenoxyacetyl)glutathione. The reactions were catalyzed by the enzyme acyl-CoA: 6-APA acyltransferase (AT) from Penicillium chrysogenum and in both cases the synthesis of antibiotics was enhanced by CoA. Penicillin K, a natural penicillin, was also synthesized "in vitro" by incubating (S-octanoyl)glutathione, 6-APA and AT, but in this case the formation of antibiotic required the presence of CoA. Furthermore, benzylpenicillin was obtained from (S-phenylacetyl)cysteinylglycine and 6-APA, suggesting that some intermediates of the gamma-glutamyl cycle are directly involved in the biosynthesis of penicillins. To explain "in vivo" formation of this beta-lactam antibiotic, a biosynthetic pathway which includes several glutathione-S-derivatives and a non-enzymatic reaction, is proposed.

Acyltransferases↗

PubMed1989

Regulation of colominic acid biosynthesis by temperature: role of cytidine 5'-monophosphate N-acetylneuraminic acid synthetase.

Synthesis of colominic acid in Escherichia coli K-235 is strictly regulated by temperature. Evidence for the role of cytidine 5'-monophospho-N-acetylneuraminic acid (CMP-Neu5Ac) synthetase in this regulation was obtained by measuring its level in E. coli grown at 20 and 37 degrees C. No activity was found in E. coli grown at 20 degrees C. CMP-Neu5Ac started to be quickly synthesized when bacteria grown at 20 degrees C were transferred to 37 degrees C and was halted when cells grown at 37 degrees C were transferred to 20 degrees C. These findings suggest that temperature regulates the synthesis of this enzyme and therefore the concentration of CMP-Neu5Ac necessary for the biosynthesis of colominic acid.

Escherichia coli↗