Cold-adapted digestive aspartic protease of the clawed lobsters Homarus americanus and Homarus gammarus: biochemical characterization.

Aspartic proteinases in the gastric fluid of clawed lobsters Homarus americanus and Homarus gammarus were isolated to homogeneity by single-step pepstatin-A affinity chromatography; such enzymes have been previously identified as cathepsin D-like enzymes based on their deduced amino acid sequence. Here, we describe their biochemical characteristics; the properties of the lobster enzymes were compared with those of its homolog, bovine cathepsin D, and found to be unique in a number of ways. The lobster enzymes demonstrated hydrolytic activity against synthetic and natural substrates at a wider range of pH; they were more temperature-sensitive, showed no changes in the K(M) value at 4°C, 10°C, and 25°C, and had 20-fold higher k(cat)/K(M) values than bovine enzyme. The bovine enzyme was temperature-dependent. We propose that both properties arose from an increase in molecular flexibility required to compensate for the reduction of reaction rates at low habitat temperatures. This is supported by the fast denaturation rates induced by temperature.

Isolation, biochemical characterization, and molecular modeling of American lobster digestive cathepsin D1.

An aspartic proteinase was isolated from American lobster gastric fluid. The purified cathepsin D runs as a single band on native-PAGE displaying proteolytic activity on a zymogram at pH 3.0, with an isoelectric point of 4.7. Appearance of the protein in SDS-PAGE, depended on the conditions of the gel electrophoresis. SDS treatment by itself was not able to fully unfold the protein. Thus, in SDS-PAGE the protein appeared to be heterogeneous. A few minute of boiling the sample in the presence of SDS was necessary to fully denature the protein that then run in the gel as a single band of ~50 kDa. The protein sequence of lobster cathepsin D1, as deduced from its mRNA sequence, lacks a 'polyproline loop' and ß-hairpin, which are characteristic of some of its structural homologues. A comparison of amino acid sequences of digestive and non-digestive cathepsin D-like enzymes from invertebrates showed that most cathepsin D enzymes involved in food digestion, lack the polyproline loop, whereas all non-digestive cathepsin Ds, including the American lobster cathepsin D2 paralog, contain the polyproline loop. We propose that the absence or presence of this loop may be characteristic of digestive and non-digestive aspartic proteinases, respectively.

Aspartic cathepsin D endopeptidase contributes to extracellular digestion in clawed lobsters Homarus americanus and Homarus gammarus.

Acid digestive proteinases were studied in the gastric fluids of two species of clawed lobster (Homarus americanus and Homarus gammarus). An active protein was identified in both species as aspartic proteinase by specific inhibition with pepstatin A. It was confirmed as cathepsin D by mass mapping, N-terminal, and full-length cDNA sequencing. Both lobster species transcribed two cathepsin D mRNAs: cathepsin D1 and cathepsin D2. Cathepsin D1 mRNA was detected only in the midgut gland, suggesting its function as a digestive enzyme. Cathepsin D2 mRNA was found in the midgut gland, gonads, and muscle. The deduced amino acid sequence of cathepsin D1 and cathepsin D2 possesses two catalytic DTG active-site motifs, the hallmark of aspartic proteinases. The putatively active cathepsin D1 has a molecular mass of 36.4 kDa and a calculated pI of 4.14 and possesses three potential glycosylation sites. The sequences showed highest similarities with cathepsin D from insects but also with another crustacean cathepsin D. Cathepsin D1 transcripts were quantified during a starvation period using real-time qPCR. In H. americanus, 15 days of starvation did not cause significant changes, but subsequent feeding caused a 2.5-fold increase. In H. gammarus, starvation caused a 40% reduction in cathepsin D1 mRNA, and no effect was observed with subsequent feeding.

Estudio de dos metodos para evitar la nefropatia asociada a contraste radiologico / Evaluation of two methods to avoid contrast associated nephropathy

En este trabajo se trata de establecer la eficacia de dos métodos para evitar la nefropatía asociada a contraste readiológico (N.A.C.). Se estudió en forma prospectiva y randomizada una muestra de 75 pacientes de los cuales 25 se asignaron al grupo Control: sin intervenciones, 25 al grupo Salina: solución salina 0.45 por ciento E.V., 1,5 cc/kg/hr, 6 horas antes y después del estudio angiográfico y 25 al grupo Dopa: igual al anterior más dopamina 2 mug/kg/min, 30 minutos antes del estudio y hasta su finalización. Se consideró como TO a la valoración realizada al momento del ingreso del paciente, T1 a las 24 y T2 a las 48 horas. En TO se registraron: edad; sexo; antecendetes patológicos; drogas y creatininemia. En T1: diuresis y creatininemia y en T2 creatininemia. Se consideró como N.A.C al aumento del 25 por ciento de la cratininemia em T2. Estadística: La edad fue analizada con Kruskal-Wallis H, las variables continuas con ANOVA y las nominales con el cálculo de X2 con corrección de Yates o Ficher según el tamaño e la muestra. La frecuencia de N.A.C en cada uno de los grupos se estudió mediante el análisis de tendencia lineal de proporciones. La N.A.C se presentó en 13/25 (OR:1) pacientes del grupo Control, 7/25 (OR 0,36) del grupo Salina y 5/25 (OR 0.23) del grupo Dopa (p=0.01). No se hallaron diferencias significativas en la diuresis ni en las cifras de creatininemia. Se concluye que la hidratación durante seis horas previas y posteriores al estudio con solución salina al 0.45 por ciento y el mismo plan con el agregado de dopamina son eficaces para prevenir la N.A.C. (AU)

Estudio de dos metodos para evitar la nefropatia asociada a contraste radiologico / Evaluation of two methods to avoid contrast associated nephropathy

En este trabajo se trata de establecer la eficacia de dos métodos para evitar la nefropatía asociada a contraste readiológico (N.A.C.). Se estudió en forma prospectiva y randomizada una muestra de 75 pacientes de los cuales 25 se asignaron al grupo Control: sin intervenciones, 25 al grupo Salina: solución salina 0.45 por ciento E.V., 1,5 cc/kg/hr, 6 horas antes y después del estudio angiográfico y 25 al grupo Dopa: igual al anterior más dopamina 2 mug/kg/min, 30 minutos antes del estudio y hasta su finalización. Se consideró como TO a la valoración realizada al momento del ingreso del paciente, T1 a las 24 y T2 a las 48 horas. En TO se registraron: edad; sexo; antecendetes patológicos; drogas y creatininemia. En T1: diuresis y creatininemia y en T2 creatininemia. Se consideró como N.A.C al aumento del 25 por ciento de la cratininemia em T2. Estadística: La edad fue analizada con Kruskal-Wallis H, las variables continuas con ANOVA y las nominales con el cálculo de X2 con corrección de Yates o Ficher según el tamaño e la muestra. La frecuencia de N.A.C en cada uno de los grupos se estudió mediante el análisis de tendencia lineal de proporciones. La N.A.C se presentó en 13/25 (OR:1) pacientes del grupo Control, 7/25 (OR 0,36) del grupo Salina y 5/25 (OR 0.23) del grupo Dopa (p=0.01). No se hallaron diferencias significativas en la diuresis ni en las cifras de creatininemia. Se concluye que la hidratación durante seis horas previas y posteriores al estudio con solución salina al 0.45 por ciento y el mismo plan con el agregado de dopamina son eficaces para prevenir la N.A.C.