RESUMO
Enzymatic properties of the enzyme 11 beta-hydroxysteroid dehydrogenase (11-HSD), which confers mineralocorticoid selectivity, have been explored in the aldosterone-sensitive collecting duct (CCD) and the aldosterone-insensitive Pars Recta (PR) of the rat kidney. After incubation of freshly isolated tubular segments with [3H]corticosterone (3H-B) or [3H]dehydrocorticosterone (3H-A), the rate of transformation of 3H-B into 3H-A (dehydrogenase activity), or the reverse reaction (reductase activity) were measured by HPLC, Vmax for dehydrogenase activity was found to be 8- to 10-fold higher in CCD than PR. The enzyme functions over a very wide range (0.1-5000 nM) of corticosterone concentration. In CCD, enzyme kinetics suggest either the presence of two 11-HSD forms, differing by their affinity for corticosterone, or complex kinetics. Addition of NAD or NADP to permeabilized tubules revealed that dehydrogenase activity is NAD-dependent in CCD and NADP-dependent in PR. Cofactor addition was ineffective in intact tubules. CCD exhibited an exclusive dehydrogenase activity, whereas in PR dehydrogenase and reductase activity were found. No regulation of dehydrogenase activity could be evidenced in adrenalectomized rats receiving or not aldosterone, corticosterone or dexamethasone, for 2 h, 3 days or 4 days. We conclude that 11-HSD in the CCD and PR differs by its Vmax and cofactor dependence. Corticosteroid hormones do not influence 11-HSD activity.
Assuntos
Homeostase , Hidroxiesteroide Desidrogenases/metabolismo , Túbulos Renais Distais/enzimologia , Túbulos Renais Proximais/enzimologia , 11-beta-Hidroxiesteroide Desidrogenases , Adrenalectomia , Aldosterona/farmacologia , Animais , Permeabilidade da Membrana Celular , Corticosterona/análogos & derivados , Corticosterona/metabolismo , Corticosterona/farmacologia , Dexametasona/farmacologia , Ativação Enzimática/efeitos dos fármacos , Feminino , Túbulos Renais Coletores/efeitos dos fármacos , Túbulos Renais Coletores/enzimologia , Cinética , NAD/farmacologia , NADP/farmacologia , Ratos , Ratos Wistar , TrítioRESUMO
N-Acetyl-beta-D-glucosaminidase (NAG) isoenzyme profile in primary cultures of rabbit kidney proximal tubule cells was studied. Confluent cells had high levels of NAG activity, but ion exchange chromatography showed that the NAG isoenzyme profile in cultured cells was different from that of rabbit renal cortex homogenates and freshly isolated cells. Confluent cultured cells contained an atypical acidic isoform, absent in homogenates and freshly isolated cells in which the predominant isoform is NAG-A (a heterodimer alpha beta). The fact that this atypical isoform was able to hydrolyse the synthetic substrate 4-methylumbelliferyl-beta-N-acetylglucosaminide-6-sulphate indicated that it probably was an alpha-subunit homodimer. These results suggest subunit rearrangement within NAG polypeptide chains linked to down-regulation of beta-subunit production in cultured rabbit proximal cells. The change in isoenzyme profile in cultured cells may make it difficult to use primary cultures of rabbit proximal tubule cells to establish correlations between in vitro and in vivo studies using NAG isoenzymes as a nephrotoxicity index, as illustrated by the effects of gentamicin.
Assuntos
Acetilglucosaminidase/metabolismo , Isoenzimas/metabolismo , Túbulos Renais Proximais/enzimologia , Animais , Antibacterianos/administração & dosagem , Antibacterianos/toxicidade , Células Cultivadas , Cromatografia por Troca Iônica , Regulação para Baixo , Gentamicinas/administração & dosagem , Gentamicinas/toxicidade , Hidrólise , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/efeitos dos fármacos , Inibidores da Síntese de Proteínas/administração & dosagem , Inibidores da Síntese de Proteínas/toxicidade , CoelhosRESUMO
D-Galacto-D-xylo-D-glucans (amyloids) from Balsamina, Tropaeolum, and Tamarindus seeds behave in a similar manner in the presence of various glycosidase preparations: slow depolymerization by enzymes from several germinated or non-germinated seeds, and hydrolysis into monosaccharides and oligosaccharides by commercial cellulase and hemicellulase preparations from fungi. A purified cellulase from Penicillium notatum gave a dialyzable fraction almost exclusively composed of alpha-D-xylopyranosyl-(1 leads to 6)-D-glucose residues and a nondialyzable fraction composed of chains of beta-D-(1 leads to 4) [With some (1 leads to 3)]-glucopyranosyl residues; beta-D-galactopyranosyl-(1 leads to 2)-alpha-D-xylosyl groups are linked to some of the beta-D-glucosyl residues at O-6. The presence of (1 leads to 3)-linkages in the D-glucan chain of the Balsamina was verified by methylation and sequential periodate oxidation-borohydride reduction; the distribution of the substituents on the D-glucan chain is not regular. The main D-glucan backbone, where the beta-D-glucosyl residues are partly linked at O-6 to beta-D-galactosyl-(1 leads to 2)-D-xylosyl groups, is linked to D-glucan chains where almost all the D-glucose units are linked at O-6 by one alpha-D-xylosyl group. The presence of 3,6-di-O-methyl-D-glucose after permethylation and hydrolysis suggests that the xyloglucan chains are linked to O-2 of the D-glucosyl units of the galactoxyloglucan backbone.
Assuntos
Amiloide , Sementes/análise , Galactose/análise , Glucose/análise , Glicosídeo Hidrolases , Especificidade da Espécie , Xilose/análiseRESUMO
Reaction of (+/-)-(3/4,5,6)-4-bromo-5,6-epoxy-3-hydroxycyclohexene with 2,3,4,6-tetra-O-acetyl-1-thio-alpha-D-glucopyranose, followed by treatment of the resulting isolated diastereoisomeric 4-bromo-3,5-dihydroxycyclohexene 1-thioglycoside derivatives with base under phase-transfer conditions, gave (R)- and (S)-(3,4,6/5)-3,4-epoxy-6-S-(1-thio-alpha-D-glucopyranosyl)-5- hydroxycyclohexene. None of them was substrate or inhibitor for cockchafer trehalase.
Assuntos
Dissacarídeos/química , Trealase/química , Alquilação , Sequência de Carboidratos , Indicadores e Reagentes , Dados de Sequência Molecular , EstereoisomerismoRESUMO
A simple diastereoselective access to amino- and hydrazinocyclopentitols is described. The key step involves a cationic rearrangement of a meso bicyclic hydrazine, followed by two successive stereoselective hydroxylations. Both racemic compounds are micromolar alpha-mannosidase (Jack bean) inhibitors.
Assuntos
Ciclopentanos/síntese química , Inibidores Enzimáticos/síntese química , Hidrazinas/química , alfa-Manosidase/antagonistas & inibidores , Técnicas de Química Combinatória , Ciclopentanos/análise , Inibidores Enzimáticos/análise , Estrutura Molecular , EstereoisomerismoRESUMO
Spiralins were purified by agarose-suspension electrophoresis after extraction with detergents from the membranes of the following spiroplasmas: Spiroplasma citri C189, S. citri Maroc (R8A2), S. citri Scaph and the honey-bee spiroplasma B88. The four proteins (molecular mass congruent to 26,000 daltons, as determined by sodium dodecyl sulphate-pore gradient electrophoresis) showed very similar amino acid compositions characterized by the absence of methionine and tryptophan and a high polarity index (greater than 49%). When compared with the amino acid composition of S. citri membrane, the four spiralins had little or no histidine, a low content of glycine, leucine, tyrosine, phenylalanine and arginine, and a high content of threonine, alanine and valine. Comparison of the amino acid compositions according to the criteria described by Cornish-Bowden (Anal. Biochem., 1980, 105, 233-238) strongly suggests that all four spiralins are related. A crossed immunoelectrophoretical comparison, however, shows that though the three proteins purified from S. citri strains (serogroup I-1) are antigenically similar, they do not seem to share common epitopes with spiralin from the honey-bee spiroplasma B88 (serogroup I-2).
Assuntos
Aminoácidos/análise , Antígenos de Bactérias/análise , Proteínas da Membrana Bacteriana Externa , Proteínas de Bactérias/análise , Epitopos/análise , Proteínas de Membrana/análise , Spiroplasma/classificação , Proteínas de Bactérias/imunologia , Membrana Celular/análise , Proteínas de Membrana/imunologia , Spiroplasma/imunologiaRESUMO
We purified angiotensin I-converting enzyme (ACE) from pig and human lung and plasma for comparison of some physicochemical properties between the endothelial membrane-bound form and the soluble form of the enzyme. After affinity chromatography on Sepharose CL-4B/lisinopril, gel-filtration HPLC on Superose 12 achieved homogeneity for both forms as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Whatever the source of ACE, the molecular weight was 300 +/- 40 kDa after calibration of Superose 12 with standard globular proteins and 172 +/- 4 kDa by SDS-PAGE, with or without reduction, a result suggesting interactions between the glycopolypeptide chain and the chromatographic gel possibly related to the overall shape and sugar content of the enzyme. Ion-exchange HPLC analysis on TSK-DEAE showed that the membrane-bound and soluble forms of ACE are not isoenzymes, although isoelectrofocusing did show that the isoelectric point of soluble ACE was lower than those of tissue ACE, suggesting a different glycosylation. No significant difference between porcine and human ACE appeared. HPLC methods seem to be of particular interest for the purification of ACE with a high yield and for the analysis of its putative differently glycosylated isoforms.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Pulmão/enzimologia , Peptidil Dipeptidase A/isolamento & purificação , Animais , Cromatografia por Troca Iônica , Glicosilação , Humanos , Ponto Isoelétrico , Peso Molecular , Peptidil Dipeptidase A/sangue , Peptidil Dipeptidase A/química , Especificidade da Espécie , SuínosRESUMO
Hexosaminidase and alkaline phosphatase activities in rabbit articular chondrocytes have been studied under different cell culture conditions. Chondrocytes were cultured in monolayer primary culture, monolayer subcultured to the fifth passage (in vitro aging) and cultured within a collagen gel; enzymatically released cartilage cells were used as control. Under these conditions, the two enzymes behave quite differently in relationship to alteration of the chondrocyte phenotype in culture. Increased lysosomal hexosaminidase activity could be considered to be a marker of the dedifferentiated phenotype in monolayer subculture; membrane alkaline phosphatase activity could be used as a marker of non-proliferating cells.
Assuntos
Fosfatase Alcalina/metabolismo , Cartilagem Articular/enzimologia , beta-N-Acetil-Hexosaminidases/metabolismo , Animais , Cartilagem Articular/citologia , Células Cultivadas , Técnicas de Cultura/métodos , Cinética , CoelhosRESUMO
1. Using derivatives or non-symmetrical analogs of alpha,alpha-trehalose, we studied the catalytic specificities of trehalases from various species: Pseudomonas fluorescens, Melolontha vulgaris, porcine and human kidneys. 2. alpha,Beta-trehalose, beta,beta-trehalose, 6,6'dideoxy alpha,alpha-trehalose, alpha-D-xylopyranosyl alpha-D-xylopyranoside were shown to be neither substrates nor inhibitors. 3. 6'deoxy alpha,alpha-trehalose, alpha-D-glucopyranosyl alpha-D-xylopyranoside, alpha-D-allopyranosyl alpha-D-glucopyranoside and alpha-D-galactosyl alpha-D-glucopyranoside, which all possess an intact alpha-D-glucopyranosyl residue, were split by all these trehalases. 4. alpha-D-glucopyranosyl alpha-D-mannopyranoside, alpha,alpha-trehalosamine are competitive inhibitors. 5. These results show the importance of the primary alcohol group at C-6, of the equatorial configuration of the OH groups at C-2, C-3 and C-4 and of the modification of the structure at C-2 of the substrate for the catalytic activity.
Assuntos
Besouros/metabolismo , Pseudomonas fluorescens/metabolismo , Suínos/metabolismo , Trealase/metabolismo , Animais , Humanos , Especificidade da Espécie , Especificidade por SubstratoRESUMO
The only specific treatments of allergy are long and exacting desensitization by subcutaneous injections of the allergens. While oral administration of allergens could greatly facilitate these treatments, effective delivery systems are needed to prevent allergen degradation in the gastrointestinal tract and to enable their uptake by Peyer's patches. The potential for bee-venom phospholipase A2 (PLA2) to be used in such oral immunotherapy was tested. For this purpose, PLA2 potential alterations were analysed when encapsulated into poly(D,L-lactide-co-glycolide) microspheres by double emulsion solvent evaporation. It was shown that microencapsulation had only limited effects on the integrity of the entrapped PLA2, which retained its fully specific murine IgE binding capacity. Thus, PLA2 loaded microspheres could represent a potential delivery system for bee venom allergy-specific oral immunotherapy.
Assuntos
Alérgenos/administração & dosagem , Venenos de Abelha/administração & dosagem , Dessensibilização Imunológica/métodos , Composição de Medicamentos/métodos , Imunoglobulina E/metabolismo , Fosfolipases A/metabolismo , Alérgenos/química , Animais , Venenos de Abelha/química , Biodegradação Ambiental , Cápsulas , Imunoglobulina E/administração & dosagem , Camundongos , Camundongos Endogâmicos , Fosfolipases A/administração & dosagem , Fosfolipases A2 , Ligação ProteicaRESUMO
The effects of gentamicin on N-acetyl-beta-D-glucosaminidase (NAG) and acid phosphatase (AcP), two lysosomal enzymes present in proximal renal tubule cells, were studied in the PKSV-PCT cell line derived from proximal convoluted tubules from the kidney of a transgenic mouse carrying SV40 large T antigen under the control of the L-type pyruvate kinase gene. Gentamicin (400 micrograms/ml for 72 hr) did not alter cell viability, but significantly reduced cell growth and favored the formation of myeloid bodies. Gentamicin (50 to 800 micrograms/ml for 72 hr) decreased in a dose-dependent manner the cellular NAG in PKSV-PCT cells and stimulated its secretion by 20 to 60%. Chloroquine (50 to 100 microns) and ammonium chloride (NH4Cl, 30mM), two lysosomotropic amines known to stimulate the secretion of lysosomal enzymes in fibroblasts and macrophages, also stimulated secreted NAG in PKSV-PCT cells. However, the effect of chloroquine was less marked in PKSV-PCT cells than in cultured mouse 3T3 fibroblasts. Gentamicin induced lysosomal alkalinization but, in contrast to chloroquine and NH4Cl, the aminoside strongly stimulated the secretion of AcP. The secretion induced by gentamicin was nonpolarized, since the percentage of secreted NAG significantly increased from both the apical and basal sides of PKSV-PCT cells grown on permeable filters. Thus, these data suggest that gentamicin alters the secretion of NAG and AcP by a non-specific pathway and indicate that the PKSV-PCT cell line is a suitable system to examine the cellular action of drugs in kidney proximal tubule cells.
Assuntos
Gentamicinas/toxicidade , Túbulos Renais Proximais/efeitos dos fármacos , Acetilglucosaminidase/metabolismo , Fosfatase Ácida/metabolismo , Cloreto de Amônio/farmacologia , Animais , Células Cultivadas , Cloroquina/farmacologia , Túbulos Renais Proximais/patologia , Túbulos Renais Proximais/fisiologia , Camundongos , Camundongos TransgênicosRESUMO
beta-methylaspartate ammonia-lyase, EC 4.3.1.2, (beta-methylaspartase) from Clostridium tetanomorphum was used to produce a 40/60 molar ratio of (2S,3R) and (2S,3S)-3-methylaspartic acids, 2a and 2b, respectively, from mesaconic acid 1 as substrate, on a large scale. To prepare (3R,4R)-3-methyl-4-(benzyloxycarbonyl)-2-oxetanone (benzyl 3-methylmalolactonate) 6, 2a and 2b were transformed, in the first step, into 2-bromo-3-methylsuccinic acids 3a and 3b and separated. After three further steps, (2S,3S)-3a yielded the alpha, beta-substituted beta-lactone (3R,4R) 6 with a very high diastereoisomeric excess (> 95% by chiral gas chromatography). The corresponding crystalline polymer, poly[benzyl beta-(2R,3S)-3-methylmalate] 8, prepared by an anionic ring opening polymerization, was highly isotactic as determined by 13C NMR. Catalytic hydrogenolysis of lactone 6 yielded (3R,4R)-3-methyl-4-carboxy-2-oxetanone (3-methylmalolactonic acid) 7, to which reactive, chiral, or bioactive molecules can be attached through ester bonds leading to polymers with possible therapeutic applications. Because of the ability of beta-methylaspartase to catalyse both syn- and anti-elimination of ammonia from (2S,3RS)-3-methylaspartic acid 2ab at different rates, the (2S,3R)-stereoisomer 2a was retained and isolated for further reactions. These results permit the use of the chemoenzymatic route for the preparation of both optically active and racemic polymers of 3-methylmalic acid with well-defined enantiomeric and diastereoisomeric compositions.