RESUMEN
We have investigated several parameters of glucocerebrosidase in cultured skin fibroblasts from patients with various clinical phenotypes of Gaucher disease. In this study no strict correlation was found between the clinical manifestations of Gaucher disease and the parameters investigated in fibroblasts. These parameters included the specific activity of the enzyme in extracts towards natural lipid and artificial substrate in the presence of different activators; the enzymic activity per unit of glucocerebrosidase protein; the rate of synthesis of the enzyme and its stability; and the post-translational processing of the enzyme. In addition, the activity in situ of glucocerebrosidase in fibroblasts was investigated using a novel method by analysis of the catabolism of NBD-glucosylceramide in cells that were loaded with bovine serum albumin-lipid complexes. Again, no complete correlation with the clinical phenotype of patients was detectable. Glucocerebrosidase in fibroblasts from most non-neuronopathic (type 1) Gaucher disease patients differs in some aspects from enzyme in cells from patients with neurological forms (types 2 and 3). The stimulation by activator protein and phospholipid is clearly more pronounced in type 1 than in types 2 and 3; the enzymic activity per unit of glucocerebrosidase protein in type 1 is severely reduced in the presence of taurocholate and the amount of glucocerebrosidase appears (near) normal in contrast to the situation in types 2 and 3 Gaucher fibroblasts. However, this distinction was not always consistent; glucocerebrosidase in fibroblasts from some type 1 Gaucher patients, particularly some South African cases, was comparable in properties to enzyme in type 2 and 3 patients.
Asunto(s)
Enfermedad de Gaucher/enzimología , Glucosilceramidasa/metabolismo , Catepsina D/metabolismo , Células Cultivadas , Centrifugación por Gradiente de Densidad , Electroforesis en Gel de Poliacrilamida , Fibroblastos/enzimología , Enfermedad de Gaucher/genética , Glucosilceramidasa/genética , Glucosilceramidas/metabolismo , Humanos , Immunoblotting , Mutación , Fenotipo , Temperatura , beta-N-Acetilhexosaminidasas/metabolismoRESUMEN
Glucocerebrosidase was purified to homogeneity from spleens of control subjects and Type 1 Gaucher disease patients by immunoaffinity chromatography. Activation of the enzyme by taurocholate, phosphatidylserine and sphingolipid activator protein 2 (saposin C; SAP-2) was investigated by titration of combinations of various effectors in the absence and presence of Triton X-100. The specific activity of Type 1 Gaucher glucocerebrosidase was found to be less than 20% of the corresponding control value under most conditions. However, in the presence of optimal amounts of activator protein SAP-2 and phosphatidylserine (and in the absence of Triton X-100 and/or taurocholate), the specific activity of mutant enzyme towards artificial and natural lipid substrates was close to normal when measured at pH 5.0-5.5. At pH values below 5.0, the specific activity of mutant enzyme decreased more rapidly compared to that of control enzyme. The activity of Type 1 Gaucher glucocerebrosidase in the intact cell might, in a comparable manner, be highly dependent on the extent of activation by endogenous activators and on the intralysosomal pH. Values for residual glucocerebrosidase activity, as measured in vitro in extracts of cells and tissues from Type 1 Gaucher disease patients, are indeed highly dependent on the assay conditions employed. Consequently such measurements are of little value in the assessment of the actual capacity for glucosylceramide hydrolysis and for prediction of the clinical severity of the disease.
Asunto(s)
Enfermedad de Gaucher/enzimología , Glucosilceramidasa/metabolismo , Bazo/enzimología , Detergentes/farmacología , Estabilidad de Enzimas , Glucosilceramidasa/genética , Glucosilceramidasa/aislamiento & purificación , Humanos , Concentración de Iones de Hidrógeno , Cinética , Mutación , Octoxinol , Fosfatidilserinas/farmacología , Polietilenglicoles/farmacología , Valores de Referencia , Ácido Taurocólico/farmacologíaRESUMEN
We investigated the peroxisomal fatty acid beta-oxidation system in liver and cultured skin fibroblasts from patients with X-linked adrenoleukodystrophy known to accumulate very long chain fatty acids. In order to examine whether the deficient peroxisomal oxidation of very long chain fatty acids in these patients results from a deficiency in one of the peroxisomal beta-oxidation enzyme proteins (acyl-CoA oxidase, bifunctional protein with enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase activities and 3-oxoacyl-CoA thiolase) we carried out immunoblotting experiments using antibodies directed against the peroxisomal beta-oxidation enzyme proteins from rat liver. Furthermore, we studied the oxidation of palmitoyl-CoA and lignoceroyl-CoA in homogenates of fibroblasts from the patients. The results indicate that the peroxisomal beta-oxidation enzyme proteins are not only present immunologically but also functionally active which suggests that the defect in X-linked adrenoleukodystrophy is, indeed, as recently suggested by Hashmi and coworkers (FEBS Lett 1986;196:247-250) at the level of a deficient peroxisomal activation of very long chain fatty acids.
Asunto(s)
Acilcoenzima A/metabolismo , Adrenoleucodistrofia/metabolismo , Esclerosis Cerebral Difusa de Schilder/metabolismo , Ácidos Grasos/metabolismo , Microcuerpos/metabolismo , Adrenoleucodistrofia/genética , Ácidos y Sales Biliares/metabolismo , Fibroblastos/metabolismo , Ligamiento Genético , Humanos , Hígado/metabolismo , Oxidación-Reducción , Cromosoma XRESUMEN
We investigated the peroxisomal beta-oxidation system in liver from a patient with clinical features similar to those in the cerebrohepatorenal (Zellweger) syndrome and with elevated levels in body fluids of very-long-chain fatty acids and intermediates in the biosynthesis of bile acids. The peroxisomal beta-oxidation of fatty acids, measured as the cyanide-insensitive formation of [14C]acetyl units from [14C]palmitoyl-CoA, was very low in the patient (less than 10% of the values in control subjects). Immunoblotting experiments using antibodies to peroxisomal beta-oxidation enzymes indicated that peroxisomal 3-oxoacyl-CoA thiolase (acyl-CoA:acetyl-CoA C-acyltransferase, EC 2.3.1.16) was deficient. Addition of purified rat-liver peroxisomal 3-oxoacyl-CoA thiolase to a reaction mixture containing liver homogenate from the patient restored peroxisomal beta-oxidation. We conclude that the deficiency of peroxisomal 3-oxoacyl-CoA thiolase is responsible for the very low peroxisomal beta-oxidation activity and for the accumulation of very-long-chain fatty acids and intermediates in the biosynthesis of bile acids. Furthermore, the finding that both very-long-chain fatty acids and abnormal bile acids accumulate in this patient suggests that a single peroxisomal 3-oxoacyl-CoA thiolase is involved in the oxidative chain shortening of both very-long-chain fatty acids and the coprostanoic acids.
Asunto(s)
Acetil-CoA C-Aciltransferasa/deficiencia , Aciltransferasas/deficiencia , Hígado/enzimología , Microcuerpos/enzimología , Acetil-CoA C-Aciltransferasa/metabolismo , Catalasa/metabolismo , D-Aminoácido Oxidasa/metabolismo , Femenino , Glutamato Deshidrogenasa/metabolismo , Humanos , LactanteRESUMEN
Antibodies raised against the soluble form of acid sphingomyelinase from human urine and placenta are able to precipitate about 70% of the sphingomyelinase activity present in preparations of urinary sphingomyelinase. In contrast, no precipitation of sphingomyelinase activity occurs in detergent-containing preparations from placenta or splenic membranes. The formation of immune complexes between the antibodies and urinary sphingomyelinase is inhibited if detergents are added. With the non-ionic detergent Triton X-100 significant inhibition occurs only above the critical micellar concentration of the detergent. With the anionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulphonate (Chaps) substantial inhibition is already observed below the critical micellar concentration of the detergent.
Asunto(s)
Detergentes/farmacología , Lisosomas/enzimología , Hidrolasas Diéster Fosfóricas/análisis , Esfingomielina Fosfodiesterasa/análisis , Tensoactivos/farmacología , Animales , Ácidos Cólicos/farmacología , Humanos , Sueros Inmunes , Técnicas de Inmunoadsorción , Octoxinol , Placenta/enzimología , Placenta/ultraestructura , Polietilenglicoles/farmacología , Conejos , Espectrofotometría , Esfingomielina Fosfodiesterasa/orina , Bazo/enzimologíaRESUMEN
A soluble form of lysosomal sphingomyelinase was partially purified from human urine using concanavalin A-Sepharose 4B, Sephadex G-100 and octyl-Sepharose 4B chromatography. The octyl-Sepharose 4B eluate was used to immunise a rabbit. The antiserum obtained was able to precipitate about 70% of the sphingomyelinase activity present in urine from control subjects. Both the immunoprecipitable and non-precipitable activities were found to be deficient in urine from patients with Niemann-Pick disease Type A and Type B. In contrast, both activities were present in urine from patients with Niemann-Pick disease Type C. The antiserum was able to precipitate about 80% of the sphingomyelinase activity present in an aqueous extract of placenta.
Asunto(s)
Anticuerpos/inmunología , Hidrolasas Diéster Fosfóricas/orina , Esfingomielina Fosfodiesterasa/orina , Animales , Antígenos/inmunología , Cromatografía , Femenino , Fibroblastos/inmunología , Humanos , Técnicas de Inmunoadsorción , Enfermedades de Niemann-Pick/enzimología , Placenta/enzimología , Embarazo , Conejos/inmunología , Esfingomielina Fosfodiesterasa/inmunología , Bazo/enzimologíaRESUMEN
The immunoblotting technique was used to identify sphingomyelinase protein in samples of tissue and urine after subjection to polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. In a sphingomyelinase preparation purified from control urine a prominent band was seen with an Mr of 28 000 Da. Glycoprotein fractions from urine and placenta, a membrane extract from spleen, and a partially purified sphingomyelinase preparation from placenta contained the 28 000-Da band plus additional, higher-Mr bands. The 28 000-Da band was detectable in urine from a patient with Niemann-Pick disease type C, but not in urine from patients with Niemann-Pick disease types A and B. It is concluded that sphingomyelinase is composed of at least one polypeptide with an Mr of 28 000 Da and that this polypeptide is deficient in the urine of patients with Niemann-Pick disease types A and B.
Asunto(s)
Enfermedades de Niemann-Pick/enzimología , Hidrolasas Diéster Fosfóricas/deficiencia , Esfingomielina Fosfodiesterasa/deficiencia , Humanos , Lisosomas/enzimología , Peso Molecular , Enfermedades de Niemann-Pick/clasificación , Esfingomielina Fosfodiesterasa/inmunología , Esfingomielina Fosfodiesterasa/orinaRESUMEN
A two-step procedure is described for the isolation of lysosomal alpha-glucosidase from human urine. In the second step, affinity chromatography on Sephadex G-100, two fractions with acid alpha-glucosidase activity were obtained. Fraction I contained alpha-glucosidase of Mr 109000, whereas fraction II contained components of Mr 76000 and 70000. alpha-Glucosidase in fraction I had an Mr similar to that of the precursor of alpha-glucosidase detected in the medium of fibroblasts after labelling with [14C]leucine. The components in fraction II had Mr identical to those of the mature forms of alpha-glucosidase found in placenta or cultured human skin fibroblasts. alpha-Glucosidase in fraction I contained mannose 6-phosphate (3.5 mol/mol polypeptide). No mannose 6-phosphate was present in the components in fraction II. Fraction I, but not fraction II, was avidly endocytosed by alpha-glucosidase-deficient cultured human skin fibroblasts. Endocytosis of fraction I was inhibited by mannose 6-phosphate. The pH optimum and Km values for p-nitrophenyl alpha-glucoside, maltose and glycogen of fractions I and II alpha-glucosidase were almost identical. However, the activity with glycogen relative to that of either p-nitrophenyl alpha-glucoside or maltose was lower in fraction I than in fraction II. It is concluded that fraction I consists of the precursor form of alpha-glucosidase and fraction II of the mature forms of the enzyme. The importance of urine as a source of precursors of lysosomal enzymes is discussed.
Asunto(s)
Precursores Enzimáticos/orina , Glucosidasas/orina , Lisosomas/enzimología , alfa-Glucosidasas/orina , Adulto , Fibroblastos/enzimología , Humanos , Inmunoquímica , Cinética , Masculino , Manosafosfatos/metabolismo , Piel/enzimologíaRESUMEN
The maturation of lysosomal alpha-glucosidase in cultured human skin fibroblasts was studied using a monoclonal antibody that distinguishes between the precursor and mature forms of the enzyme. Monoclonal antibodies against alpha-glucosidase isolated from placenta were produced by the hybridoma technique [Hilkens et al. (1981) Biochim. Biophys. Acta 678, 7-11]. One of these monoclonal antibodies, that synthesized by clone 43G8, reacts with the mature forms, but not with the precursor form of alpha-glucosidase isolated from urine. By means of pulse-labelling studies, it could be shown that monoclonal antibody 43G8 does not react with either the intracellular or the secreted precursor of alpha-glucosidase from cultured human skin fibroblasts. However, the antibody does react with the intermediate and mature forms of alpha-glucosidase. Endocytosis of the precurosor of alpha-glucosidase from urine by fibroblasts is followed by its conversion to a form with lower molecular mass. After endocytosis urinary precursor alpha-glucosidase is converted to a form that binds to monoclonal antibody 43G8. The t 1/2 for this conversion is 2 h. The conversion is inhibited by addition of leupeptin to the culture medium. It is concluded that a thiol proteinase is involved in the maturation of alpha-glucosidase in fibroblasts and the appearance of the antigenic determinant for 43G8.