RESUMEN

When colonizing the digestive tract of mono-associated rats, Ruminococcus gnavus E1 - a bacterium isolated from human faeces - produced a trypsin-dependent anti-Clostridium perfringens substance collectively named Ruminococcin C (RumC). RumC was isolated from the caecal contents of E1-monocontaminated rats and found to consist of two antimicrobial fractions: a single peptide (RumCsp) of 4235 Da, and a mixture of two other peptides (RumCdp) with distinct molecular masses of 4324 Da and 4456 Da. Both RumCsp and RumCdp were as effective as metronidazole in combating C. perfringens and their activity spectra against different pathogens were established. Even if devoid of synergistic activity, the combination of RumCsp and RumCdp was observed to be much more resistant to acidic pH and high temperature than each fraction tested individually. N-terminal sequence analysis showed that the primary structures of these three peptides shared a high degree of homology, but were clearly distinct from previously reported amino acid sequences. Amino acid composition of the three RumC peptides did not highlight the presence of any Lanthionine residue. However, Edman degradation could not run beyond the 11th amino acid residue. Five genes encoding putative pre-RumC-like peptides were identified in the genome of strain E1, confirming that RumC was a bacteriocin. This is the first time that a bacteriocin produced in vivo by a human commensal bacterium was purified and characterized.

Asunto(s)

Proteínas Bacterianas/biosíntesis , Bacteriocinas/biosíntesis , Clostridium perfringens/metabolismo , Intestinos/microbiología , Ruminococcus/metabolismo , Alanina/análogos & derivados , Alanina/química , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/química , Bacteriocinas/química , Bacteriocinas/genética , Ciego/microbiología , Clostridium perfringens/crecimiento & desarrollo , Genes Bacterianos , Calor , Concentración de Iones de Hidrógeno , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Ratas , Sulfuros/química

RESUMEN

Canavan disease is an autosomal-recessive neurodegenerative disorder caused by a lack of aspartoacylase, the enzyme that degrades N-acetylaspartate (NAA) into acetate and aspartate. With a view to studying the mechanisms underlying the action of human aspartoacylase (hASP), this enzyme was expressed in a heterologous Escherichia coli system and characterized. The recombinant protein was found to have a molecular weight of 36 kDa and kinetic constants K(m) and k(cat) of 0.20 +/- 0.03 mM and 14.22 +/- 0.48 s(-1), respectively. Sequence alignment showed that this enzyme belongs to the carboxypeptidase metalloprotein family having the conserved motif H(21)xxE(24)(91aa)H(116). We further investigated the active site of hASP by performing modelling studies and site-directed mutagenesis. His21, Glu24 and His116 were identified here for the first time as the residues involved in the zinc-binding process. In addition, mutations involving the Glu178Gln and Glu178Asp residues resulted in the loss of enzyme activity. The finding that wild-type and Glu178Asp have the same K(m) but different k(cat) values confirms the idea that the carboxylate group contributes importantly to the enzymatic activity of aspartoacylase.

Asunto(s)

Amidohidrolasas/química , Amidohidrolasas/metabolismo , Enfermedad de Canavan/enzimología , Amidohidrolasas/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Secuencia de Bases , Enfermedad de Canavan/genética , Dominio Catalítico/genética , ADN Complementario/genética , Escherichia coli/genética , Ácido Glutámico/química , Histidina/química , Humanos , Técnicas In Vitro , Cinética , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Peso Molecular , Mutagénesis Sitio-Dirigida , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Zinc/metabolismo

RESUMEN

High-performance anion exchange chromatography coupled with a pulsed amperometric detection system (HPAEC-PAD) was used to evaluate the extent of chemical hydrolysis of three fructooligosaccharides (FOS) including 1-kestose (beta-D-Fru-(2-->1)(2)-alpha-D-glucopyranoside, GF2), nystose (beta-D-Fru-(2-->1)(3)-alpha-D-glucopyranoside, GF3), and fructofuranosylnystose (beta-D-Fru-(2-->1)(4)-alpha-D-glucopyranoside, GF4). A kinetic study was carried out at 80, 90, 100, 110, and 120 degrees C in aqueous solutions buffered at pH values of 4.0, 7.0, and 9.0. Under each experimental condition, the determination of the respective amounts of reactants and hydrolysis products showed that FOS hydrolysis obeyed pseudo-first-order kinetics as the extent of hydrolysis, which decreased at increasing pH values, increased with temperature. The three oligomers were found to be degraded mainly under acidic conditions, and at the highest temperature value (120 degrees C), a quick and complete acid degradation of each FOS was observed. Using the Arrhenius equation, rate constants, half-life values, and activation energies were calculated and compared with those obtained from sucrose under the same experimental conditions. It appeared that the hydrolysis of FOS took place much more easily at acidic pH than at neutral or basic pH values.

Asunto(s)

Oligosacáridos/metabolismo , Temperatura , Tampones (Química) , Cromatografía Líquida de Alta Presión , Concentración de Iones de Hidrógeno , Hidrólisis , Cinética , Minerales , Soluciones , Sacarosa/metabolismo

RESUMEN

Fructans are food-grade non-digestible carbohydrates that exert beneficial nutritional effects. Their characterization and quantification is required for food-labeling purposes. We describe the suitability of high-performance anion-exchange chromatography coupled with pulsed amperometric detection for the identification and quantification of fructans in fresh fruits (various apple and pear cultivars, plum, banana) as well as in commercial stewed fruits obtained from a local manufacturer. After extraction with water and appropriate filtration, inulobiose [beta-D-Fru-(2-->1)-beta-D-fructofuranoside; F2], 1-kestose [beta-D-Fru-(2-->1)2-alpha-D-glucopyranoside; GF2] and nystose [beta-D-Fru-(2-->1)3-alpha-D-glucopyranoside; GF3] were completely separated in a single 36-min run using a Dionex CarboPac PA 100 column and the new quadruple-potential waveform, originally tailored for oligosaccharide separation. No measurable amounts of F3 and GF4 were detected within the group of studied fruit products. Peak identification was realized using standards. The method is easy, reproducible, and sensitive since as little as 28 microg of sugar per gram dry matter can be quantified. Banana and plum are the varieties containing the highest levels of fructans (about 6000 microg per gram dry matter). The maturity of the fruit appears to have a great influence on the level of GF2. Samples of apple-banana stewed fruits contained the highest total fructan concentration (about 700 microg per gram dry matter). Accurate quantification of fructans will allow more precise nutritional formulation and diet selection for higher fructan consumption.

Asunto(s)

Cromatografía Líquida de Alta Presión/métodos , Cromatografía por Intercambio Iónico/métodos , Electroquímica/métodos , Fructanos/análisis , Frutas/química , Estudios de Evaluación como Asunto

RESUMEN

An accurate carbohydrate analysis method, namely high-performance anion-exchange chromatography with pulsed amperometric detection was successfully applied to the study of sucrose hydrolysis under enzymatic (baker's yeast invertase) conditions. The hydrolysis was monitored by determining sucrose degradation and the corresponding formation of D-glucose, D-fructose and five intermediate fructans using a CarboPac PA-100 (Dionex) analytical anion-exchange column. Highly reproducible results were obtained. The unknown fructans were collected from a semi-preparative CarboPac PA-100 (Dionex) column, neutralized and then desalted on a column containing mixed bed resin AG 501-X8 (D) before identification of the chemical structure. This procedure permitted us to obtain about 20 microg of pure product which is not enough for NMR analysis. Detailed GC-MS analytical data of the methylated compounds indicated that these oligosaccharides were beta-D-Fru-(2 --> 1)-beta-D-Fru-(2 --> 1)-alpha-D-glucopyranoside (1-kestose), beta-D-Fru-(2 --> 6)-alpha-D-glucopyranoside (6-beta fructofuranosylglucose), beta-D-Fru-(2 --> 1)-beta-D-fructofuranoside (inulobiose), beta-D-Fru-(2 --> 6)-beta-D-Fru-(2 --> 1)-alpha-D-glucopyranoside (6-kestose) and beta-D-Fru-(2 --> 6)-alpha-D-Glc-(1 --> 2)-beta-D-fructofuranoside (neokestose) coeluating with a disaccharide.

Asunto(s)

Cromatografía Líquida de Alta Presión/métodos , Cromatografía por Intercambio Iónico/métodos , Enzimas/química , Sacarosa/química , Electroquímica , Cromatografía de Gases y Espectrometría de Masas , Hidrólisis , Espectroscopía de Resonancia Magnética , Reproducibilidad de los Resultados

RESUMEN

The amino acid sequence analysis of the human and porcine aminoacylases-1, the carboxypeptidase S precursor from Saccharomyces cerevisiae, the succinyl-diaminopimelate desuccinylase from Escherichia coli, Haemophilus influenzae and Corynebacterium glutamicum, the acetylornithine deacetylase from Escherichia coli and Dictyostelium discoideum and the carboxypeptidase G(2) precursor from Pseudomonas strain, using the Basic Local Alignment Search Tool (BLAST) and the Position-Specific Iterated BLAST (PSI-BLAST), allowed us to suggest that all these enzymes, which share common functional and biochemical features, belong to the same structural family. The three amino acid blocks which were found to be highly conserved, using the CLUSTAL W program, could be assigned to the catalytic active site, based on the general three-dimensional structure of the carboxypeptidase G(2) from the Pseudomonas strain precursor. Six additional proteins with the same signature have been retrieved after performing two successive PSI-BLAST iterations using the sequence of the conserved motif, namely Lactobacillus delbrueckii aminoacyl-histidine dipeptidase, Streptomyces griseus aminopeptidase, Saccharomyces cerevisiae aminopeptidase Y precursor, two Bacillus stearothermophilus N-carbamyl-L-amino acid amidohydrolases and Pseudomonas sp. hydantoin utilization protein C. The three conserved amino acid motifs corresponded to the following blocks: (i) [S, G, A]-H-x-D-x-V; (ii) G-x-x-D; and (iii) x-E-E. This new sequence signature is clearly different from that commonly reported in the literature for proteins belonging to the ArgE/DapE/CPG2/YscS family.

Asunto(s)

Amidohidrolasas/química , Proteínas Bacterianas , Metaloendopeptidasas/química , Amidohidrolasas/metabolismo , Secuencia de Aminoácidos , Animales , Bacterias/enzimología , Secuencia de Consenso , Bases de Datos Factuales , Humanos , Proteínas de la Membrana/química , Metaloendopeptidasas/metabolismo , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/química , Saccharomyces cerevisiae/enzimología , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Porcinos

RESUMEN

Baker's yeast invertase was found to catalyse transfructosylation reactions in aqueous and anhydrous organic media with sucrose as a substrate, leading to the formation of five intermediate fructans in addition to the release of D-glucose (D-Glc)and D-fructose (D-Fru). All the reaction products were separated and quantitatively estimated using high performance anion exchange-pulsed amperometric detection equipment. The unknown products were subsequently identified by linkage analysis as beta-D-Fru-(2 --> 1)-beta-D-Fru-(2 --> 1)- alpha-D-glucopyranoside (1-kestose), beta-D-Fru- (2 --> 6)-alpha-D-glucopyranoside (6-beta-fructofuranosylglucose), beta-D-Fru-(2 -->1) -beta-D-fructofuranoside (inulobiose), beta-D-Fru-(2 --> 6)-beta-D-Fru-(2 --> 1)-alpha-D-glucopyranoside (6-kestose) and beta-D-Fru-(2 --> 6)-alpha-D-Glc-(1 --> 2)-beta-D-fructofuranoside (neokestose); and this last was eluted together with a disaccharide. The time-course of sucrose hydrolysis via fructan production in 2 ml of a 50 mM sodium acetate buffer (pH 4.5) containing 0.2 M sucrose and 25 U of invertase was different from that in 2 ml of anhydrous toluene with 1.46 M sucrose and 1,000 U of invertase as a suspended powder. Under the latter experimental conditions, invertase was found to exhibit cyclic behaviour, where sucrose was degraded and subsequently synthesised. This observation has not yet been reported, as far as we know.

Asunto(s)

Biotecnología/métodos , Fructanos/biosíntesis , Glicósido Hidrolasas/metabolismo , Saccharomyces cerevisiae/enzimología , Sacarosa/metabolismo , Cromatografía por Intercambio Iónico/métodos , Medios de Cultivo , Hidrólisis , beta-Fructofuranosidasa

RESUMEN

The cDNA encoding Aspergillus niger cinnamoyl esterase (FAEA) with its native signal sequence was isolated by reverse transcriptase-polymerase chain reaction, sequenced, and expressed in Pichia pastoris. Secretion yields up to 300 mg l(-1) were obtained in buffered medium. The recombinant FAEA was purified to homogeneity using a one-step purification protocol and found to be identical to the native enzyme with respect to size, pI, immunoreactivity and N-terminal sequence. Specific activity, pH and temperature optimum, and kinetic parameters were also found similar to the native esterase. FAEA is thus the first fungal esterase efficiently produced using a heterologous system.

Asunto(s)

Aspergillus niger/enzimología , Hidrolasas de Éster Carboxílico/genética , Hidrolasas de Éster Carboxílico/metabolismo , Pichia/enzimología , Proteínas Recombinantes/metabolismo , Aspergillus niger/genética , Biotecnología/métodos , Hidrolasas de Éster Carboxílico/química , Ácidos Cumáricos/metabolismo , ADN Complementario/genética , Cinética , Pichia/genética , Pichia/crecimiento & desarrollo , ARN de Hongos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN

RESUMEN

The soluble acylase I from rat kidney was purified to homogeneity using a five-step procedure. As the resulting protein was found to have a relative molecular mass of 125 kDa based on size-exclusion chromatography and 44 kDa based on SDS/PAGE, the native protein was taken to consist of three subunits. The amino-acid sequence of a peptide resulting from limited proteolysis of the polypeptide chain with proteinase K, which was determined by microsequencing (RHEFHALRAGFALDEGLA), was found to be very similar to the corresponding sequence of porcine kidney acylase I. However, as N-furyl-acryloyl-L-methionine, a synthetic substrate for porcine acylases, was not hydrolyzed by the rat enzyme, it was suggested that the polypeptide chain might differ in other respects from those of the other acylases I. A full length cDNA coding for the rat kidney acylase I was therefore isolated and found to contain a 1224-bp open reading frame encoding a protein consisting of 408 amino-acid residues, which corresponded to a calculated molecular mass of 45 847 Da per subunit. The deduced amino-acid sequence showed 93.6% and 87.2% identity with that of the human liver and porcine kidney, respectively.

Asunto(s)

Amidohidrolasas/genética , Amidohidrolasas/metabolismo , Riñón/enzimología , Amidohidrolasas/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Endopeptidasa K/metabolismo , Humanos , Masculino , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Mapeo Peptídico , Ratas , Ratas Wistar , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Porcinos

RESUMEN

Efficient production of recombinant Aspergillus niger family 11 1, 4-beta-xylanase was achieved in Pichia pastoris. The cDNA-encoding XylA fused to the Saccharomyces cerevisiae invertase signal peptide was placed under the control of the P. pastoris AOX1 promoter. Secretion yields up to 60 mg/liter were obtained in synthetic medium. The recombinant XylA was purified to homogeneity using a one-step purification protocol and found to be identical to the enzyme overexpressed in A. niger with respect to size, pI, and immunoreactivity. N-terminal sequence analysis of the recombinant protein indicated that the S. cerevisiae signal peptide was correctly processed in P. pastoris. The purified protein has a molecular weight of 19,893 Da, in excellent agreement with the calculated mass, and appears as one single band on isoelectric focusing with pI value around 3.5. Electrospray ionization mass spectrometry confirmed the presence of one major isoform produced by P. pastoris and the absence of glycosylation. The recombinant enzyme was further characterized in terms of specific activity, pH profile, kinetic parameters, and thermostability toward birchwood xylan as substrate and compared with the xylanase purified from A. niger. Both enzymes exhibit a pH optimum at 3.5 and maximal activity at 50 degrees C. The enzyme activity follows normal Michaelis-Menten kinetics with K(m) and V(max) values similar for both enzymes. P. pastoris produced recombinant xylanase in high yields that can be obtained readily as a single form. A. niger xylanase is the first microbial xylanase efficiently secreted and correctly processed by P. pastoris.

Asunto(s)

Aspergillus niger/química , Proteínas Fúngicas/aislamiento & purificación , Xilosidasas/aislamiento & purificación , Secuencia de Aminoácidos , Western Blotting , Electroforesis en Gel de Poliacrilamida , Endo-1,4-beta Xilanasas , Estabilidad de Enzimas , Proteínas Fúngicas/biosíntesis , Proteínas Fúngicas/genética , Calefacción , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Pichia/enzimología , Pichia/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Xilosidasas/biosíntesis , Xilosidasas/genética

RESUMEN

CNBr fragments from porcine intestinal glycerol-ester hydrolase were separated by SDS/PAGE under reducing and nonreducing conditions, and their amino-acid sequences were analysed. Two intra-chain disulfide bridges were identified, namely Cys70-Cys99 (loop A) and Cys256-Cys267 (loop B). As the Cys71 sulfhydryl group could not be alkylated with iodoacetamide, it is suggested that the residue is blocked rather than being present in the free form. The two disulfide bridges of intestinal glycerol-ester hydrolase are present in the cholinesterase family, although the enzyme showed only about 35% identity with these proteins. Furthermore, the finding that glycerol-ester hydrolase was partly inactivated under reducing conditions suggests that one or both disulfide bridges are important for the enzyme conformation. Lastly, glycerol-ester hydrolase was also found to hydrolyse cholinergic substrates, although residues Trp86 and Asp74 which are considered to be the main constituents of the 'anionic' subsite responsible for substrate binding in cholinesterases were absent from loop A. Other amino-acid residues in the glycerol-ester hydrolase may therefore be responsible for the binding of cholinergic substrates to the enzyme.

Asunto(s)

Hidrolasas de Éster Carboxílico/química , Disulfuros/química , Mucosa Intestinal/enzimología , Animales , Sitios de Unión , Hidrolasas de Éster Carboxílico/metabolismo , Colinesterasas/química , Bromuro de Cianógeno/química , Cisteína/análisis , Ditiotreitol/farmacología , Estabilidad de Enzimas , Yodoacetamida/farmacología , Lipasa/metabolismo , Fragmentos de Péptidos/química , Mapeo Peptídico , Unión Proteica , Conformación Proteica , Análisis de Secuencia , Relación Estructura-Actividad , Especificidad por Sustrato , Porcinos

RESUMEN

Crystal structures of human pancreatic alpha-amylase (HPA) in complex with naturally occurring inhibitors have been solved. The tetrasaccharide acarbose and a pseudo-pentasaccharide of the trestatin family produced identical continuous electron densities corresponding to a pentasaccharide species, spanning the -3 to +2 subsites of the enzyme, presumably resulting from transglycosylation. Binding of the acarviosine core linked to a glucose residue at subsites -1 to +2 appears to be a critical part of the interaction process between alpha-amylases and trestatin-derived inhibitors. Two crystal forms, obtained at different values of pH, for the complex of HPA with the protein inhibitor from Phaseolus vulgaris (alpha-amylase inhibitor) have been solved. The flexible loop typical of the mammalian alpha-amylases was shown to exist in two different conformations, suggesting that loop closure is pH-sensitive. Structural information is provided for the important inhibitor residue, Arg-74, which has not been observed previously in structural analyses.

Asunto(s)

Inhibidores Enzimáticos/metabolismo , Lectinas/metabolismo , Páncreas/enzimología , Lectinas de Plantas , Proteínas de Plantas/metabolismo , Polisacáridos/metabolismo , alfa-Amilasas/química , alfa-Amilasas/metabolismo , Acarbosa/química , Acarbosa/metabolismo , Acarbosa/farmacología , Amino Azúcares/metabolismo , Amino Azúcares/farmacología , Arginina/química , Arginina/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Electrones , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Glicosilación , Humanos , Enlace de Hidrógeno , Concentración de Iones de Hidrógeno , Lectinas/química , Lectinas/farmacología , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas de Plantas/química , Proteínas de Plantas/farmacología , Polisacáridos/química , Polisacáridos/farmacología , Conformación Proteica , Trisacáridos/química , Trisacáridos/metabolismo , Trisacáridos/farmacología , alfa-Amilasas/antagonistas & inhibidores

RESUMEN

The interaction between glucose and essential amino acids at 100 degrees C at pH values ranging from 4.0 to 12.0 was investigated by monitoring the disappearance of glucose and amino acids as well as the appearance of brown color. Lysine was the most strongly destroyed amino acid, followed by threonine which induced very little additional browning as compared with that undergone by glucose. Around neutrality, the nonenzymatic browning followed pseudo-zero-order kinetics after a lag time, while the glucose and amino acid losses did not follow first-order kinetics at any of the pH values tested. Glucose was more strongly destroyed than all of the essential amino acids, the losses of which are really small at pH values lower than 9.0. However, glucose was less susceptible to thermal degradation in the presence of amino acids, especially at pH 8.0 with threonine and at pH 10.0 with lysine. The contribution of the caramelization reaction to the overall nonenzymatic browning above neutrality should lead to an overestimation of the Maillard reaction in foods.

Asunto(s)

Aminoácidos Esenciales/química , Glucosa/química , Culinaria , Concentración de Iones de Hidrógeno , Cinética , Reacción de Maillard

RESUMEN

The distribution of acylase I and acylpeptide hydrolase along the hog small intestine was investigated. No significant changes in their respective specific activity was found when the intestine was cut off and divided into eight segments (taken every 200 cm) so as to specifically study the duodenum, jejunum and ileum. Upon performing subcellular fractionation of hog enterocytes, it was observed that acylpeptide hydrolase is a soluble enzyme, while acylase I is essentially a soluble protein accounting for only 5% of the activity associated with the whole membrane fraction. The membrane-bound acylase I was neither solubilized by phosphatidylinositol-specific phospholipase C from Bacillus cereus nor by detergents which are commonly used to solubilize alkaline phosphatase, a glycosylphosphatidylinositol-anchored protein. When a phase separation was carried out in Triton X-114, all the anchored-membrane proteins of the intestinal membranes were located in the detergent-rich phase, while acylase I was present in the detergent-poor phase. Finally, the immunolabeling of intestinal cells with specific antibodies definitively established the cytoplasmic localization of acylase I. Acylpeptide hydrolase and acylase I therefore both are located in the enterocyte cytoplasm.

Asunto(s)

Amidohidrolasas/metabolismo , Sistema Digestivo/enzimología , Exopeptidasas/metabolismo , Péptido Hidrolasas , Animales , Duodeno/enzimología , Íleon/enzimología , Inmunohistoquímica , Mucosa Intestinal/enzimología , Yeyuno/enzimología , Microvellosidades/enzimología , Fracciones Subcelulares/enzimología , Porcinos , Distribución Tisular

RESUMEN

The carboxypeptidase activity occurring in hog intestinal mucosa is apparently due to two distinct enzymes which may be responsible for the release of basic COOH-terminal amino acids from short peptides. The plasma membrane-bound carboxypeptidase activity which occurs at neutral optimum pH levels was found to be enhanced by CoCl(2) and inhibited by guanidinoethylmercaptosuccinic acid, o-phenanthroline, ethylenediamine tetraacetic acid and cadmium acetate; whereas the soluble carboxypeptidase activity which occurs at an optimum pH level of 5.0 was not activated by CoCl(2) and only slightly inhibited by o-phenanthroline, ethylenediamine tetraacetic acid, NiCl(2) and CdCl(2). The latter activity was presumably due to lysosomal cathepsin B, which is known to be present in the soluble fraction of hog intestinal mucosa. Although the membrane-bound enzyme was evenly distributed along the small intestine, it was not anchored in the phospholipidic bilayer via a glycosyl-phosphatidylinositol moiety, as carboxypeptidase M from human placenta is. The enzyme was not solubilized by phosphatidylinositol-specific phospholipase C, but was solubilized to practically the same extent by several detergents. The purified trypsin-solubilized form is a glycoprotein with a molecular mass of 200 kDa, as determined by performing SDS-PAGE and gel filtration, which differs considerably from the molecular mass of human placental carboxypeptidase M (62 kDa). It was found to cleave lysyl bonds more rapidly than arginyl bonds, which is not so in the case of carboxypeptidase M, and immunoblotting analysis provided further evidence that hog intestinal and human placental membrane-bound carboxypeptidases do not bear much resemblance to each other. Since the latter enzyme has been called carboxypeptidase M, it is suggested that the former might be carboxypeptidase D, the recently described new member of the carboxypeptide B-type family.

Asunto(s)

Carboxipeptidasas/metabolismo , Mucosa Intestinal/enzimología , Aminoácidos/análisis , Animales , Carboxipeptidasas/antagonistas & inhibidores , Carboxipeptidasas/aislamiento & purificación , Membrana Celular/enzimología , Detergentes , Endopeptidasas/metabolismo , Activación Enzimática , Mucosa Gástrica/enzimología , Concentración de Iones de Hidrógeno , Membranas Intracelulares/enzimología , Solubilidad , Fracciones Subcelulares/enzimología , Porcinos , Tripsina , Fosfolipasas de Tipo C/metabolismo

RESUMEN

Acylpeptide hydrolase was purified to homogeneity from porcine intestinal mucosa using a seven-step procedure including ammonium sulfate precipitation, gel filtration as well as anion exchange and affinity chromatography. The specific activity of the enzyme reached 105000 nmol/mg protein per min and the purification was as high as 5500-fold. This tetrameric enzyme is composed of four apparently identical subunits, the molecular mass of which was estimated to be 75 kDa, based on the results of amino acid analysis and gel electrophoresis performed under denaturing conditions. It is likely that the NH(2)-terminal residue may be acetylated, while serine was found to be the COOH-terminal residue. The hydrolytic activity of the enzyme toward N-acetyl-L-alanine p-nitroanilide at the optimum pH value was increased twofold in the presence of the chloride anion. The K(m) value calculated from the kinetics of the hydrolysis of acetylalanyl peptides was found to be 0.7+/-0.1 mM, whereas the V(max) values decreased from 200 to 50 nmol/min per microgram of enzyme, depending on the peptidic chain lengths. The V(max) value of the synthetic substrate (250 nmol/min per microgram of enzyme) was 25-500% higher than those of the acetylalanyl peptides, depending on the peptide chain length, although the enzyme affinity was slightly lower (1.8 mM as compared with 0.7 mM). In line with data on other animal species and on various tissues, the enzyme seemed likely to be a serine protease, since it was readily inhibited by diisopropyl fluorophosphate and diethyl pyrocarbonate. A 2377-nucleotide long cDNA coding for the enzyme was isolated from pig small intestine. The deduced amino acid sequence consisted of 731 residues and showed a single different amino acid with that of the porcine liver APH, except the N-terminal amino acid which is still probably lacking.

Asunto(s)

Mucosa Intestinal/enzimología , Péptido Hidrolasas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN Complementario/química , Electroforesis en Gel de Poliacrilamida , Inhibidores Enzimáticos/farmacología , Cinética , Hígado/enzimología , Datos de Secuencia Molecular , Péptido Hidrolasas/química , Péptido Hidrolasas/aislamiento & purificación , Porcinos

RESUMEN

A full-length cDNA clone coding for porcine pancreatic preprocarboxypeptidase A1 (prePCPA1) was isolated from a cDNA library. The open reading frame (ORF) of the nucleotide sequence was 1260 nt in length and encoded a protein of 419 amino acids (aa). The cDNA included a short signal peptide of 16 aa and a 94 aa-long activation segment. The calculated molecular mass of the mature proenzyme was 45561 Da, in accordance with that of the purified porcine pancreatic PCPA1. The deduced aa sequence of the corresponding enzyme differed from that predicted by the three-dimensional structure by 40 aa, and showed 85% identity and 55% identity to that of procarboxypeptidases A1 and A2, respectively. Moreover the sequence was identical to that of several independent cDNA clones, suggesting that it is the major transcribed gene. No evidence for a second variant was observed in the cDNA library and PCPA2 is apparently absent from the porcine pancreas. The cDNA was expressed in Saccharomyces cerevisiae under the control of the yeast triose phosphate isomerase promoter. The signal peptide of the PCPA protein efficiently directed its secretion into the culture medium (1.5 mg.L-1) as a protein of the predicted size. The recombinant proenzyme was analyzed by immunological and enzymological methods. Its activation behavior was comparable with that of the native form and led to a 35-kDa active enzyme.

Asunto(s)

Carboxipeptidasas/genética , Precursores Enzimáticos/genética , Páncreas/enzimología , Animales , Carboxipeptidasas/biosíntesis , Carboxipeptidasas A , Clonación Molecular , ADN Complementario/genética , Activación Enzimática , Precursores Enzimáticos/biosíntesis , Expresión Génica/genética , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/genética , Señales de Clasificación de Proteína/genética , Proteínas Recombinantes/genética , Saccharomyces cerevisiae/genética , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Porcinos , Tripsina/metabolismo

RESUMEN

A cDNA library was constructed in a Uni-ZAP XR vector using mRNA isolated from porcine pancreas. A full-length alpha-amylase cDNA was obtained using a combination of library screening and nested polymerase chain reaction. Sequencing of the clone revealed a 1536-nucleotide (nt) open reading frame encoding a protein of 496 amino acid (aa) residues with a signal peptide of 15 aa. The calculated molecular mass of the enzyme was 55354 Da, in accordance with those of the purified porcine pancreatic alpha-amylase forms (PPAI and PPAII) as determined by mass spectrometry. A comparison of the deduced aa sequence with published peptidic sequences of PPAI identified a number of mismatches. The sequence of the cDNA reported here provides a sequence reference for PPA in excellent agreement with the refined three-dimensional structures of both PPAI and PPAII. No evidence for a second variant was found in the cDNA library and it is most likely that PPAI and PPAII are two forms of the same protein. The primary structure of PPA shows high homology with human, mouse and rat pancreatic alpha-amylases. The 304-310 region, corresponding to a mobile loop involved in substrate binding and processing near the active site, is fully conserved.

Asunto(s)

Páncreas/enzimología , alfa-Amilasas/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN Complementario/biosíntesis , ADN Complementario/química , Biblioteca de Genes , Isoenzimas/química , Datos de Secuencia Molecular , Porcinos , alfa-Amilasas/biosíntesis

RESUMEN

A glycerol-ester hydrolase was purified to homogeneity from porcine intestinal mucosa using a partial delipidation method and an eight-step purification procedure. The isolation scheme used gave a 483-fold purification, resulting in a pure enzyme with a specific activity on tributyrin of 290 micromol x min(-1) x mg(-1). The molecular mass of the enzyme was estimated at 240 kDa, based on the results of size-exclusion chromatography, and at 60 kDa, as determined by SDS/PAGE analysis. The isoelectric focusing data obtained indicated that only one isoform with a pI of 5.1 was present. Complete identity was found to exist between the N-terminal sequence of the first 25 amino acid residues and that of a porcine liver carboxylesterase. A full-length cDNA coding for the enzyme was isolated from pig small intestine. We observed that the corresponding protein originally named intestinal glycerol-ester hydrolase definitely belongs to the carboxylesterase family. The deduced amino acid sequence consisted of 565 residues and showed 97% identity with that of porcine liver carboxylesterase and more than 50% identity with those of other carboxylesterases from different mammalian species.

Asunto(s)

Hidrolasas de Éster Carboxílico/química , Intestinos/enzimología , Monoacilglicerol Lipasas/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Carbohidratos/análisis , Carboxilesterasa , Clonación Molecular , ADN Complementario , Datos de Secuencia Molecular , Peso Molecular , Monoacilglicerol Lipasas/química , Monoacilglicerol Lipasas/genética , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Porcinos

RESUMEN

A rapid method is proposed for isolating the two main components of human pancreatic alpha-amylase (HPA I and HPA II). The isoelectric point of HPA I (7.2), the main component, was determined using an isoelectrofocusing method and found to differ from that of HPA II (6. 6). The molecular mass of HPA I (55862+/-5 Da) and that of HPA II (55786+/-5 Da) were determined by performing mass spectrometry and found to be quite similar to that of the protein moiety calculated from the amino acid sequence (55788 Da), which indicates that the human amylase is not glycosylated. The structure of both HPA I and HPA II was further investigated by performing limited proteolysis. Two fragments with an apparent molecular mass of 41 kDa and 14 kDa were obtained by digesting the isoforms with proteinase K and subtilisin, whereas digestion with papain yielded two cleaved fragments with molecular masses of 38 kDa and 17 kDa. Proteinase K and subtilisin susceptible bonds are located in the L8 loop (A domain), while the papain cut which occurs in the presence of the calcium chelator EDTA is in the L3 loop (B domain). The kinetics of the inhibition of HPA I and HPA II by acarbose, a drug used to treat diabetes and obesity, were studied using an amylose substrate. The Lineweaver-Burk primary plots of HPA I and HPA II, which did not differ significantly, indicated that the inhibition was of the mixed non-competitive type. The secondary plots gave parabolic curves. All in all, these data provide evidence that two acarbose molecules bind to HPA. In conclusion, apart from the pI, no significant differences were observed between HPA I and HPA II as regards either their molecular mass and limited proteolysis or their kinetic behavior. As was to be expected in view of the high degree of structural identity previously found to exist between human and porcine pancreatic amylases, the present data show that the inhibitory effects of acarbose on the kinetic behavior of these two amylases are quite comparable. In particular, the process of amylose hydrolysis catalyzed by HPA as well as by PPA in both cases requires two carbohydrate binding sites in addition to the catalytic site.

Asunto(s)

Páncreas/enzimología , alfa-Amilasas/química , Acarbosa , Electroforesis en Gel de Poliacrilamida , Inhibidores Enzimáticos/farmacología , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/química , Isoenzimas/aislamiento & purificación , Cinética , Peso Molecular , Trisacáridos/farmacología , alfa-Amilasas/antagonistas & inhibidores , alfa-Amilasas/aislamiento & purificación