Characterization of digestive enzymes from captive Brazilian flounder Paralichthys orbignyanus / Caracterização de enzimas digestivas do linguado brasileiro de cativeiro Paralichthys orbignyanus

Abstract Knowledge of specific enzyme activity, along with animal habits and digestive capacity is essential in formulating an appropriate diet for any species. In this study, we evaluated and characterized the activity of digestive enzymes present in the liver, intestine, and stomach of Paralichthys orbignyanus. The effects of pH and temperature on enzyme activity were also evaluated via the use of specific substrates. The use of specific substrates and inhibitors showed strong evidence of the presence of trypsin (BApNA= 0.51 ± 0.2 mU mg-1), chimotrypsin (SApNA= 2.62 ± 1.8 mU mg-1), and aminopeptidases (Leu-p-Nan =0.9709 ± 0.83 mU mg-1) in the intestine. Optimum pH for the activity of trypsin, chemotrypsin, leucino aminopeptidase, amilase, and pepsin were 9.5, 9.0, 8.0, 7.5, and 3.5, respectively, while optimum temperatures were 50, 50, 50, 40, and 45 °C, respectively. These results provide additional information regarding the biology of Brazilian flounder and can be used as a basis for further studies regarding fish feeding physiology.

Resumo O conhecimento da atividade enzimática é essencial para formular uma correta dieta específica para espécie, além de estarem correlacionadas com o hábito da alimentação e capacidade digestive. Neste estudo determinamos e caracterizamos a atividade enzimática presente no intestino, estômago e fígado do linguado Paralichthys orbignyanus. Os efeitos da temperatura e pH sobre a atividade enzimática também foram avaliados utilizando substratos específicos. O uso de substratos e inibidores específicos mostrou uma forte evidência da presença da tripsina (BApNA = 0,51 ± 0,2 mU mg-1), quimotripsina (SAPNA = 2,62 ± 1,8 mU mg-1), e as aminopeptidases (Leu-p-Nan = 0,97 ± 0,83 mU mg-1) no intestino. O pH ótimo observado para a atividade de tripsina, quimotripsina, leucino aminopeptidase, amilase e pepsina foi 9,5, 9,0, 8,0, 7,5 e 3,5, respectivamente. A temperatura ótima observada foi 50, 50, 50, 40 e 45 °C, respectivamente. Estes resultados fornecem informações adicionais sobre a biologia do linguado brasileiro e pode ser usado como base para novos estudos sobre fisiologia alimentar.

Desenvolvimento de um modelo preditivo para identificação de perda de estabilidade e ocorrência de proteólise em leite UAT / Development of a predicative model for identification of loss of stability and proteolysis occurrence in UHT milk

Este trabalho se propôs a desenvolver um modelo preditivo para identificação de perda de estabilidade e de sedimentação em leite UAT por determinação da atividade enzimática de aminopeptidase no leite por espectrofotometria. Foram analisadas amostras de leite cru, pasteurizado e UAT após envase durante seis meses, na região Sul do Brasil. Acidez, crioscopia, gordura, extrato seco total, extrato seco desengordurado e densidade foram analisados nos leites cru e pasteurizado. Amostras de leite cru foram ainda submetidas à análise de contagem de psicrotróficos e à atividade de aminopeptidase, e amostras de leite UAT estocadas foram analisadas quanto ao grau de proteólise mediante análises sensoriais e atividade de aminopeptidase. Alterações sensoriais foram observadas em tempos de estocagem menores para amostras originadas de leite cru com contagem de psicrotróficos acima de 107 UFC mL-1. Não houve correlação entre a atividade de aminopeptidase e proteólise e também não foi observada correlação significativa entre os parâmetros físico-químicos e a ocorrência de proteólise no leite estocado. O modelo estudado não foi apto para predizer perda de estabilidade e ocorrência de proteólise no leite UAT.(AU)

The aim of this work was to develop a predictive model for identifying loss of stability and sedimentation in UHT milk by determining the enzymatic activity of aminopeptidasis in milk by spectrophotometry. Samples of raw milk, pasteurized and UHT after filling for 6 months in Southern Brazil were analyzed. Acidity, freezing point, fat, total solids, nonfat solids and density were analyzed in raw and pasteurized milk. Raw milk samples were also subjected to psychrotrophic count analysis and aminopeptidasis activity and UAT samples of stored milk were analyzed for degree of proteolysis through sensory analysis and aminopeptidasis activity. Sensory changes were observed in smaller storage time for samples of raw milk originated with psychrotrophic count above 107 CFU ml-1. There was no correlation between aminopeptidasis activity and proteolysis and there was also no significant correlation between physicochemical parameters and the occurrence of proteolysis in stored milk. The model was unable to predict loss of stability and occurrence of proteolysis in UHT milk.(AU)

Molecular cloning, expression and characterization of a serine proteinase from Japanese edible mushroom, Grifola frondosa: solving the structure - function anomaly of a reported aminopeptidase

The N-terminal amino acid sequence of an aminopeptidase from Japanese edible mushroom, Grifola frondosa, was reported to have high similarity with that of a serine proteinase from basidiomycete, Agaricus bisporous (Nishiwaki and Hayashi, 2001). The full-length cDNA and the corresponding genomic DNA of the enzyme were cloned, based on the reported N-terminal amino acid sequence. The predicted open reading frame (ORF) of the cloned cDNA, encoding a product of 379 amino acids, was expressed in E. coli using pET expression vector. The expressed pro-enzyme (40 kDa) underwent autolysis to produce the mature protein (30 kDa) and a pro-peptide (10 kDa). The mature protein and the pro-peptide remained tightly bound to each other and could not be separated by Ni-NTA metal affinity chromatography or Q-Sepharose ion-exchange chromatography. The enzyme was inactive in the bound form. Upon treatment with subtilisin, the bound pro-peptide was further hydrolyzed and a high serine proteinase activity was recovered. No aminopeptidase activity was detected at any stage of the protein processing. These results clearly indicated that the N-terminal amino acid sequence and the function of the reported aminopeptidase were not derived from the same protein entity and hence caused the structure-function anomaly.

Genetic characterization of the peptidases of Polistes versicolor (Hymenoptera: Vespidae)

Starch gel electrophoresis with L-leucyl-beta-naphthylamide as substrate revealed five aminopeptidases in extracts of Polistes versicolor. These enzymes are presumably products of five structural gene loci. All but Lap aminopeptidases exhibited differential distribution in the developmental stages and in the tissues. Five dipeptidases were revealed with different dipeptides. These enzymes exhibited significant differences in their substrate preferences, but a more homogeneous distribution throughout ontogenetic developmental stages than did aminopeptidases. Electrophoretic variants of Lap4 and PepA² were detected and although a low intralocus heterozygosity was found due to the low frequency of these variants, phenotypical segregation observed at these loci in pupae extracts of some colonies points to the occurrence of more than one egg-laying female. Otherwise, the detection of Lap4 allozyme restricted to nests of one area suggests low dispersion ability of the adults of Polistes versicolor

Efeito da autólise de culturas lácticas na proteólise do queijo Prato / Effect of autolysis from lactic culture on Prato cheese proteolyis

Nesta pesquisa, estudaram-se as variações ocorridas na relação entre autólise de culturas lácticas e o desenvolvimento da proteólise de queijo Prato produzido em quatro regiões brasileiras: Santa Catarina (Queijo A), Goiás (Queijo B), São Paulo (Queijo C) e Minas Gerais (Queijo D). A análise quantitativa da população de bactérias lácticas durante a maturação mostrou perfis microbiológicos similares para todas as amostras de queijos examinadas. Após 5 dias de maturação, lactococos e estreptococos estavam presentes em números mais elevados do que lactobacilos mesofílicos e termofílicos, leuconostoc e fermentadores de lactato. Contudo, essas populações aumentaram consideravelmente no final do processo de maturação...

Purification and partial characterization of Phaseolus vulgaris seed aminopeptidase

The aminopeptidase activity of Phaseolus vulgaris seeds was measured using L-Leu-p-nitroanilide and the L-aminoacyl-ß-naphthylamides of Leu, Ala, Arg and Met. A single peak of aminopeptidase activity on Leu-ß-naphthylamide was eluted at 750 µS after gradient elution chromatography on DEAE-cellulose of the supernatant of a crude seed extract. The effluent containing enzyme activity was applied to a Superdex 200 column and only one peak of aminopeptidase activity was obtained. SDS-polyacrylamide gel electrophoresis (10 per cent) presented only one protein band with molecular mass of 31 kDa under reducing and nonreducing conditions. The aminopeptidase has an optimum pH of 7.0 for activity on all substrates tested and the highest Vmax/KM ratio for L-Leu-ß-naphthylamide. The enzyme activity was increased 40 per cent by 0.15 M NaCl, inhibited 94 per cent by 2.0 mM Zn2+, inhibited 91 per cent by sodium p-hydroxymercuribenzoate and inhibited 45 per cent by 0.7 mM o-phenanthroline and 30 µM EDTA. Mercaptoethanol (3.3 mM), dithioerythritol (1.7 mM), Ala, Arg, Leu and Met (70 µM), p-nitroaniline (0.25 mM) and ß-naphthylamine (0.53 mM) had no effect on enzyme activity when assayed with 0.56 mM of substrate. Bestatin (20 µM) inhibited 18 per cent the enzyme activity. The aminopeptidase activity in the seeds decayed 50 per cent after two months when stored at 4oC and room temperature. The enzyme is leucyl aminopeptidase metal- and thiol group-dependent.

Influencia del ácido oleico sobre la actividad angiotensinasa de astroglía cortical de rata / Influence of oleic acid in the angiotensinase activity in the cortical astroglia in rats

Objetivo. Las dietas elevadas en grasa están asociada con el desarrollo de enfermedades cerebrovasculares de distinto tipo. Distintos estudios epidemiológicos ha demostrado que la población de los países mediterráneos tienen menor porcentaje de enfermedades cerebro vasculares que la población del norte de Europa o de Estados Unidos de Norteamérica. En los países mediterráneos, la dieta habitual contiene una gran proporción de ácidos grasos monoinsaturados, mientras que en los países mas industrializados, el componente graso está constituido principalmente por grasas saturadas. Las aminopeptidasas (AP) son especialmente importantes por que están implicadas en el metabolismo y regulación de hormonas circulantes y péptidos biológicamente activos de numerosos tejidos. Algunas aminopeptidasas están relacionadas con el metabolismo de las angiotensinas en el sistema renina-angiotensina (RAS), y por tanto participan en la regulación de la presión sanguínea, tanto a nivel sistémico como local. El sistema nervioso es, probablemente, uno de los órganos mas sensibles a las alteraciones de la presión arterial. El presente trabajo se ha diseñado para estudiar el comportamiento de la actividad angiotensinasa en astrocitos de corteza frontal de rata, cultivados en presencia de distintas concentraciones de ácido oleico en el medio de cultivo. Material y métodos. La actividad angiotensinasa (aspartato-glutamato-aminopeptidasa) se analizó en cultivos primarios de astrocitos de rata, utilizando b -naftilamidas como sustratos. Resultados y conclusiones. Las disminuciones observadas en la actividad aminopeptidasa ponen de manifiesto la posible influencia del ácido oleico sobre algunos de los factores implicados en la regulación del flujo sanguíneo local o la homeostasis de líquidos y electrólitos locales en el sistema nervioso. Se discute que esta acción puede ser debida a la modificación de los procesos de comunicación intercelular mediados por uniones de tipo GAP o bien, a la modulación de los sistemas de comunicación intracelular mediados por el fosfatidilinositol y posterior activación de la proteinkinasa C, entre otros

Comparación de la tinción de gram con la detección de prolina aminopeptidasa para el diagnóstico de vaginosis bacteriana / Comparison of the gram stain with the detection of proline aminopeptidase for the diagnosis of bacterial vaginosis

La vaginosis bacteriana es la causa más frecuente de infección vaginal en mujeres en edad fértil. Se asocia con complicaciones frecuentes tanto en Obstetricia como en Ginecología, por lo que es deseable tener un test de diagnóstico rápido y exacto. La tinción de Gram de la secreción vaginal tiene buena correlación con el diagnóstico clínico de vaginosis bacteriana y actualmente es ampliamente utilizado para el diagnóstico de laboratorio. La detección de prolina aminopeptidasa en el fluido vaginal se correlaciona satisfactoriamente con el diagnóstico clínico y con la tinción de Gram. El objetivo de este estudio fue comparar la eficacia de la detección de la enzima prolina aminopeptidasa en comparación con la tinción de Gram para el diagnóstico de vaginosis bacteriana en 70 embarazadas con signos o síntomas de infección urogenital, atendidas en el Servicio de Obstetricia y Ginecología del Hospital San Borja Arriarán. Los valores correspondientes de sensibilidad y especificidad del test enzimático en comparación con la tinción de Gram fueron 78,8 por ciento y 91, 9 por ciento respectivamente. El ensayo de la enzima prolina aminopeptidasa resultó un simple y específico test para la detección de la vaginosis bacteriana pero por su baja sensibilidad en relación a la tinción de Gram no se recomienda como única técnica de diagnóstico

Basic aminopeptidase from rabbit kidney: purification and partial characterization

The aminopeptidase activity of a homogenate of rabbit kidney treated with Triton X-100 was measured using L-aminoacyl-2-naphthylatmides (AA-NA). After gradient elution ion-exchange chromatography, four peaks of aminopeptidase activity were eluted. The enzyme eluted at 450 muS containing 33.5 per cent of the activity towards Arg-NA was applied to a Superdex 75 column and presented only one protein band on 10 per cent SDS-polyacrylamide gel electrophoresis. This enzyme has an apparent molecular mass of 78 kDa, is five-fold activated by 0.15 M NaCl and the highest Vmax/Km ratio was obtained with Arg-NA. Enzyme activity was inhibited 100 per cent by 0.13 mM sodium p-hydroxymercuribenzoate, 20 per cent by 0.75 mM EDTA and 100 per cent by 0.66 mM ophenanthroline. Puromycin and bestatin behaved like competitive inhibitors with a Ki of 0.60 mM and 5.0 muM, respectively.

Anticuerpos monoclonales en la evaluación de enzimas del ribete estriado yeyunal de lactantes con diarrea persistente / Monoclonal antibodies in the assessment of the jejunal striatum border enzymes in infants with longstanding diarrhea

En las biopsias de mucosa yeyunal de 10 pacientes con diarrea persistente se estudió expresión de las enzimas lactasa, sacarasa-isomaltasa, maltasa y aminopeptidasa, del ribete estriado, mediante anticuerpos monoclonales y los resultados se contrastaron con los síntomas y signos clínicos, morfológicos (microscopía de luz), actividad disacaridásica (Dahlqvist) y la expresión de lactasa por un método histoquímico. Se obtuvo expresión de aminopeptidasa en criptas y vellosidades, mediante los anticuerpos correspondientes. La expresión por anticuerpos histoquímica y actividad enzimática (Dahlqvist) fueron concordantes para la expresión de lactasa en las vellosidades, mientras en las criptas se registró positividad sólo en 2 casos. En las vellosidades los anticuerpos monoclonales tendieron a producir más reacciones positivas para sacarasa-isomaltasa en los casos con menos daño morfológico, excepto en uno de deficiencia primaria; en las criptas el resultado fue positivo en todos, menos dos pacientes, en los que tampoco hubo positividad en las vellosidades. Los anticuerpos monoclonales pueden aportar información útil para entender mejor los mecanismos de daño y reparación de las enzimas del ribete estriado y estimar el pronóstico de la lesión

Padronização e variações do teste da atividade da prolina aminopeptidase: novo método de diagnóstico das vaginoses bacterianas / Padronization and variations of the proline aminopeptidase activity tests: a new method of a bacterial vaginosis diagnosis

A vaginose bacteriana (VB) pode ser diagnosticada clinicamente e através de métodos laboratoriais que utilizem a microscopia óptica convencional (exame a fresco, método de Gram e Papanicolaou), cultura e análises cromatográficas. O teste da atividade da prolina aminopeptidase (TAPA) é uma técnica que envolve uma reação enzimática e foi testada com o objetivo de esclarecer os casos de diagnósticos duvidosos ou indeterminados para substituir a cultura e as análises cromatográficas que são técnicas morosas e dispendiosas. O TAPA foi comparado com a cultura semi-quantitativa para Gardnerella vaginalis demonstrando valor “p” significativo, sensibilidade igual a 95%, especificidade igual a 78% e valor preditivo positivo igual a 96%.

Structural and immunological studies of GPI-anchored brush border hydrolases

The complement of brush border hydrolases provides an excellent model system for study of the structure, topology and assembly of plasma membrane proteins. Among the peptidases of the renal brush border are a number of glycosylphosphatidylinositol (GPI)-anchored proteins, especially membrane dipeptidase and aminopeptidase P. Affinity purification protocols have led to the isolation of homogeneous forms of these proteins and membrane dipeptidase has been cloned and expressed in Xenopus oocytes and Cos-1 cells. The core glycan structures of both human and porcine dipeptidase have been determined, allowing direct comparisons of the GPI anchors on the same protein in different species. Aminopeptidase P has been compared in the brush borders of pig kidney and intestine and may well be anchored in distinct ways in the two tissues. Immunological cross-reactivity of polyclonal antibodies to these two proteins has revealed the phospholipase C-cleaved GPI anchor as the common epitope and defined those components of the anchor important for recognition. These antibodies are also proving useful in characterizing GPI-derived mediators that may be involved in cell signalling processes. These abundant ectopeptidases offer a number of advantages for studies of the biochemistry of mammalian GPI anchors

Inhibition of aminopeptidase activity by aromatic and other cyclic compounds

The effect of 2-naphthylamine, p-nitroaniline, o-phenanthroline, sodium deoxycholate and hydrocortisone succinate on the activity of human urine aminopeptidase, rat kidney methionyl and arginyl aminopeptidase, soybean and Enterolobium contortisiliquum seed aminopeptidase was studied using aminoacyl-2-naphthylamide and L-Leu-p-nitroanilide as substrates. Ki values ranged from 10 microM to 2.7 mM. On the basis of Ki and Km values, and catalytic efficiency for each enzyme, it is clear that the aminopeptidases from human urine and from soybean seed should be assayed with both substrates, whereas L-Leu-p-nitroaniline is a more appropriate substrate for the rat kidney aminopeptidases. Sodium deoxycholate is a better inhibitor than hydrocortisone succinate. Non-competitive inhibition was observed in all cases except for E. contortisiliquum seed aminopeptidase

Purification and partial characterization of Enterolobium contortisiliquum seed arylamidase

Arylamidase activity was isolated from Enterolobium contortisiliquum seeds (2 U/g) using L-Leu-2-naphthlamide as substrate to monitor the prification. The enzyme preparation was purified 733-fold by ammonium sulfate precipitation, and by ion eschange and gel filtration chromatography, in 6,6% yield. SDS-Polyacrylamide gel electrophoresis after fast protein liquid chromatography on a Mono Q column, showed only one protein band with a molecular weight of 35 kDa. The optimum pH for arylamidase activity was 6.5. Taking the hydrolysis rate of Lys-2-naphthylamide as one, the relative rates at which the other substrates were hydrolyzed were: Leu-2-naphthlamide, 30, Met-2-naphthlamide, 18, Arg-2-naphthlamide, 2, Ala-2-naphthylamide, 1.5, and L-Leu-p-nitroanilide, 26. The arylamidase activity was inhibited 50% by 0.1 mM HgCl2, 0.1 mM ZnCl2, 0.13 mM NiCl2, 0.2 mM o-phenanthroline and 1 * M soidum p-hydroxymercuribenzoate, and activated 35% by 5.0 * M EDTA. Iodoacetate (0.067 mM), dithioerythritol and 2-mercaptoethanol (3.3 mM), and chloride ions (0.2 M) had no effect on the enzyme activity

Alanilaminopeptidasa urinaria: valor diagnóstico en pacientes con trasplante renal en dependencia del criterio de decisión / Urinary alanylaminopeptidase: diagnostic value in patients with renal transplantation depending on decision criterium

Se estudió el valor diagnóstico de la alanilaminopeptidasa urinaria en pacientes con transplante renal, en dependencia del criterio de decisión indicativo de un aumento en su excreción. Los mejores resultados se obtienen cuando la excreción diaria de esta enzima es normalizada en forma de promedios de tres días consecutivos y tiene lugar en aumento igual o superior a el 20% en comparación con el dia anterior

Método de punto final para la determinación de la actividad urinaria de la alanilaminopeptidasa / The end-point method for the determination of urinary activity of alanylaminopeptidase

Se desarrolló un método de punto final para la determinación de la actividad urinaria de la alanilaminopeptidasa a partir de un método cinético. Una concentración 0,5 M de HCl resultó eficaz para detener la reacción enzimática. Con un tiempo de incubación de 15 minutos se logra compensar la disminución de la sensibilidad debido a la adición del ácido. En estas condiciones los resultados entre el método cinético y el nuestro son similares. Este hecho unido a la alta precisión, avalan la introducción de este método en los laboratorios de nuestro país para la detección de daño renal de diferente causa

Partial purification and properties of rat urine arylamidases

Arylamidases were isolated from rat urine using L-aminoacyl-2-naphthylamides and L-Leu-p-nitroanilide as substrates to monitor the purification. Ion-exchange chromatography separated three peaks of activity (A, B and C). after gel filtration chromatography, the second and third peaks (B and C) were further purified to provide B1 and C1. Each behaved like a single active protein band on 7.5% polyacrylamide gel electrophoresis without SDS. he molecular weights of fractions B1 and C1 determined by SDS-PAGE were 440 and 270 kDa, respectively. The pH optimum for arylamidase activity was 7.5 for both forms on all substrate for both forms. The arylamidase activity of B1 and C1 was not affected by the presence of chloride ions and was increased in the presence of CaCl2 and MnCl2 only when L-Glu-2-naphthylamide was used as substrate. EDTA (3.3-33.0 micronM) and o-phenanthroline (0.1-1.0mM) but not -SH(0.08-0.67 mM) or -S-S-(0.42-3.3mM) group reagents inhibited the arylamidase activity. Hydrolysis of L-Leu-2-naphthylamide by fractions B1 and C1 was competitively inhibited by leucine (0.14-0.56 mM), indomethacin and puromycin (67-267 micronM) and bestatin (8.3-33.3 micronM). For each inhibitor, the Ki values were similar in the two fractions: 100 micronM for L-leucine, 10 micronM for indomethacin and puromycin and 1.0 micronM for bestatin. The enzymatic properties of fractions B1 and C1 were similar to those reported for fraction A1 by Alves et al. (Brazilian Journal of Medical and Biological...