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1.
Sci Total Environ ; 667: 41-48, 2019 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-30825820

RESUMEN

Experiments have shown that increasing dissolved CO2 concentrations (i.e. Ocean Acidification, OA) in marine ecosystems may act as nutrient for primary producers (e.g. fleshy algae) or a stressor for calcifying species (e.g., coralline algae, corals, molluscs). For the first time, rapid habitat dominance shifts and altered competitive replacement from a reef-forming to a non-reef-forming biogenic habitat were documented over one-year exposure to low pH/high CO2 through a transplant experiment off Vulcano Island CO2 seeps (NE Sicily, Italy). Ocean acidification decreased vermetid reefs complexity via a reduction in the reef-building species density, boosted canopy macroalgae and led to changes in composition, structure and functional diversity of the associated benthic assemblages. OA effects on invertebrate richness and abundance were nonlinear, being maximal at intermediate complexity levels of vermetid reefs and canopy forming algae. Abundance of higher order consumers (e.g. carnivores, suspension feeders) decreased under elevated CO2 levels. Herbivores were non-linearly related to OA conditions, with increasing competitive release only of minor intertidal grazers (e.g. amphipods) under elevated CO2 levels. Our results support the dual role of CO2 (as a stressor and as a resource) in disrupting the state of rocky shore communities, and raise specific concerns about the future of intertidal reef ecosystem under increasing CO2 emissions. We contribute to inform predictions of the complex and nonlinear community effects of OA on biogenic habitats, but at the same time encourage the use of multiple natural CO2 gradients in providing quantitative data on changing community responses to long-term CO2 exposure.


Asunto(s)
Biodiversidad , Dióxido de Carbono/análisis , Ecosistema , Invertebrados/fisiología , Agua de Mar/análisis , Animales , Italia , Mar Mediterráneo , Modelos Biológicos , Dinámicas no Lineales , Océanos y Mares , Caracoles/fisiología
2.
Biochim Biophys Acta ; 1119(3): 239-46, 1992 Mar 12.
Artículo en Inglés | MEDLINE | ID: mdl-1547268

RESUMEN

Studies were conducted to investigate the mechanism by which acidic phospholipid-containing vesicles stimulate purified placental glucosylceramidase activity towards the water-soluble substrate 4-methylumbelliferyl-beta-D-glucopyranoside (MUGlc). Vesicles composed of pure phosphatidic acid (PA) or pure phosphatidylserine (PS) stimulated the activity of the enzyme about 20-fold. The inclusion of cholesterol and phosphatidylcholine (PC), beside PA, into the vesicles slightly improved their stimulatory effect. Further addition of oleic acid (OA) markedly increased the stimulation (50-fold). By ultracentrifugation and gel permeation procedures it was shown that, under optimal conditions for stimulation of the MUGlc hydrolysis by acidic phospholipid-containing vesicles, purified glucosylceramidase spontaneously binds to their surface. Interestingly, the molar fraction of the acidic phospholipid into the mixed vesicles, rather than its concentration in the assay, is the crucial parameter for activation and binding of the enzyme. The importance of glucosylceramidase association with appropriate vesicles for enzyme activation was indicated by observing that the presence of 0.2 M citrate-phosphate buffer (pH 5.5), that prevented the binding to PA-containing surfaces, also inhibited the enzyme activity. Our results indicate that the reconstitution of glucosylceramidase activity occurs through the spontaneous tight association of the enzymatic protein with preformed acidic phospholipid-containing vesicles.


Asunto(s)
Glucósidos/metabolismo , Glucosilceramidasa/metabolismo , Himecromona/análogos & derivados , Liposomas/metabolismo , Fosfolípidos/metabolismo , Centrifugación por Gradiente de Densidad , Cromatografía , Concentración de Iones de Hidrógeno , Himecromona/metabolismo
3.
Biochim Biophys Acta ; 1047(1): 57-62, 1990 Oct 22.
Artículo en Inglés | MEDLINE | ID: mdl-2248964

RESUMEN

We have found that, under some experimental conditions, the placental glucosylceramidase shows an anomalous behaviour on gel filtration chromatography. At pH 5.6, the optimal pH of the enzymatic assay, the purified enzyme remains bound to either Superose 6 or TSK-40-XL HPLC columns, while the interaction of the crude glucosylceramidase contained in the water extract of the lysosome-mitochondrial fraction of placenta with the two HPLC gel matrices is much weaker. The quite different behaviour of the crude compared to the purified enzyme may be explained by the formation in the crude preparation of associated form(s) of glucosylceramidase with suitable endogenous compound(s), which compete with the gel matrices for the binding to the enzyme. The most likely one component of the enzyme complex is the placental activating factor, previously reported by us (Vaccaro et al. (1985) Biochim. Biophys. Acta 836, 157-166), as indicated by the negligible stimulation of the crude enzyme activity on addition of the factor, either before or after passage through the HPLC columns. On the assumption that the behaviour of crude glucosylceramidase on gel filtration becomes similar to that of the purified enzyme when its interaction with endogenous substance(s) is impaired, we have identified some conditions which prevent the formation of the enzyme associated form(s): (a) the addition of guanidine chloride (0.2 M), a cahotropic agent, to the crude preparation; and (b) the increase of pH up to 8. In conclusion, taking advantage of the anomalous behaviour of glucosylceramidase on gel filtration chromatography, evidence has been obtained that placental glucosylceramidase may occur under several forms which had not been previously reported; a difference in experimental conditions can promote the formation of one or another form, by possibly affecting the composition and/or the stoichiometry and/or the stability of the enzyme complex.


Asunto(s)
Glucosilceramidasa/análisis , Placenta/enzimología , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Femenino , Glucosilceramidasa/metabolismo , Guanidina , Guanidinas/farmacología , Humanos , Concentración de Iones de Hidrógeno , Lisosomas/enzimología , Microsomas/enzimología , Placenta/ultraestructura , Embarazo
4.
Biochim Biophys Acta ; 1033(1): 73-9, 1990 Jan 29.
Artículo en Inglés | MEDLINE | ID: mdl-2302413

RESUMEN

Optimal enzymatic hydrolysis of glucosylceramide inserted into liposomes has been obtained when both acidic phospholipids and the appropriate fatty acids were added to glucosylceramide-containing liposomes. In fact, the stimulation of glucosylceramidase by acidic phospholipids was synergistically enhanced by fatty acids, whose effect was dependent upon chain length and increased on unsaturation. By following the partition of glucosylceramidase between the aqueous phase and the liposome-associated state with a flotation procedure, it has been found that phosphatidic acid (PA) and oleic acid (OA), as representatives of acidic phospholipids and activating fatty acids, respectively, were both required not only for optimal glucosylceramidase activity, but also for a tight binding of the enzyme to the liposomes. The binding was significantly less effective in the absence of either PA or OA. In the absence of both PA and OA no physical interaction between the enzyme and the liposomes was observed. Under all conditions, the glucosylceramidase activity directly correlated with the enzyme binding to the substrate-containing liposomes. Additionally, we have obtained evidence that the site(s) of the enzyme involved in the binding to the liposomes is distinct from the catalytic site; in fact, the enzyme could still associate with liposomes containing PA and OA but devoid of glucosylceramide, while it was incapable of binding to glucosylceramide-containing liposomes in the absence of PA and OA. In conclusion, the presence in liposomes of acidic phospholipids together with the appropriate fatty acids plays a key role in promoting the binding of glucosylceramidase. Consequently, when glucosylceramide is also included in the liposomes, its hydrolysis is markedly enhanced by these acidic lipids.


Asunto(s)
Cerebrósidos/metabolismo , Glucosidasas/metabolismo , Glucosilceramidasa/metabolismo , Glucosilceramidas/metabolismo , Liposomas/metabolismo , Ácidos Oléicos/farmacología , Ácidos Fosfatidicos/farmacología , Sitios de Unión , Fenómenos Químicos , Química Física , Humanos , Concentración de Iones de Hidrógeno , Ácido Oléico , Unión Proteica , Ultracentrifugación
5.
Biochim Biophys Acta ; 836(2): 157-66, 1985 Sep 11.
Artículo en Inglés | MEDLINE | ID: mdl-4027262

RESUMEN

An endogenous, heat-stable and pronase-sensitive activator for enzymatic hydrolysis of glucosylceramide was detected in the crude lysosome-mitochondria fraction of human placenta. Its properties differ distinctly in several important respects from those of the previously described glucosylceramidase activator. The activator reported here had no effect on crude glucosylceramidase with either glucosylceramide or 4-methylumbelliferyl-beta-D-glucopyranoside as the substrate in the presence of either sodium taurocholate or phosphatidylserine. On the contrary, glucosylceramide hydrolysis by the enzyme partially purified through Octyl-Sepharose 4B chromatography was stimulated by this activator 6-9-fold in the presence of either sodium taurocholate or phosphatidylserine. The Km for glucosylceramide in the presence of the activator was 1/3 of that without the activator. In the crude enzyme fraction, the activator was present in a 16-fold excess over the minimum amount necessary for full activation of the enzyme. Hydrolysis of the fluorogenic substrate by the post-Octyl-Sepharose enzyme, however, was not stimulated by the activator. Similarly, hydrolysis of galactosylceramide by galactosylceramidase obtained from the same Octyl-Sepharose chromatography was not stimulated. Our observations are consistent with the idea that glucosylceramidase is saturated by, or perhaps tightly associated with, this activator in the placenta and that they are dissociated by the Octyl-Sepharose chromatography. In fact, the properties of the combined post-Octyl-Sepharose enzyme and activator closely mimic those of the crude enzyme without added activator.


Asunto(s)
Glucosidasas/metabolismo , Glucosilceramidasa/metabolismo , Placenta/metabolismo , Proteínas Gestacionales/aislamiento & purificación , Cromatografía en Agarosa , Activación Enzimática , Femenino , Humanos , Concentración de Iones de Hidrógeno , Hidrólisis , Cinética , Lisosomas/metabolismo , Mitocondrias/metabolismo , Placenta/enzimología , Proteínas Gestacionales/fisiología , Sefarosa/análogos & derivados
6.
Biochim Biophys Acta ; 1149(1): 55-62, 1993 Jun 18.
Artículo en Inglés | MEDLINE | ID: mdl-8318531

RESUMEN

The influence of phosphatidylserine (PS) liposome size on their capacity to activate and bind purified glucosylceramidase was investigated. Gel filtration and flotation experiments showed that large unilamellar vesicles (LUV) of either pure PS or PS in admixture with phosphatidylcholine (PC) are unable to tightly bind purified glucosylceramidase, and thus, to fully stimulate its activity. By contrast, small unilamellar vesicles (SUV) of PS adsorb glucosylceramidase can either be favoured or inhibited by factors affecting the bilayer curvature of PS liposomes. An increase of PS vesicle size induced by a fusogenic agent such as poly(ethylene glycol) (PEG), decreased enzyme binding and activity. On the contrary, the reduction of PS LUV size by sonication increased their stimulating ability. Enzyme association with PS SUV is reversible. In fact, glucosylceramidase bound to PS SUV was released from the lipid surface when the SUV were transformed into larger vesicles by PEG; dissociation from the vesicles resulted in a dramatic decrease of enzyme activity. Although PS LUV are unable to reconstitute glucosylceramidase, their association with oleic acid (OA) promotes the interaction with glucosylceramidase. This phenomenon is best explained in terms of OA-induced surface defects of PS LUV, with consequent exposure of the more hydrophobic part of the membrane and hence the improved binding of hydrophobic region/s of glucosylceramidase. Our data indicate that the physical organization of the PS-containing liposomes is of critical importance of glucosylceramidase reconstitution. The observation that physical changes of the lipid surface can markedly affect the enzyme activity offers a new approach to the study of glucosylceramidase regulation.


Asunto(s)
Glucosilceramidasa/metabolismo , Membrana Dobles de Lípidos/metabolismo , Liposomas/metabolismo , Fosfatidilserinas/metabolismo , Activación Enzimática , Humanos , Membrana Dobles de Lípidos/química , Liposomas/química , Ácido Oléico , Ácidos Oléicos/farmacología , Tamaño de la Partícula
7.
FEBS Lett ; 472(1): 17-21, 2000 Apr 21.
Artículo en Inglés | MEDLINE | ID: mdl-10781797

RESUMEN

The reconstitution of the activity of the lysosomal enzyme glucosylceramidase requires anionic phospholipids and, at least, a protein factor, saposin C (Sap C). We have previously proposed a mechanism for the glucosylceramidase activation [Vaccaro et al. (1993) FEBS Lett. 336, 159-162] which implies that Sap C promotes the association of the enzyme with anionic phospholipid-containing membranes, thus favoring the contact between the enzyme and its lipid substrate, glucosylceramide. We have further investigated the properties of Sap C using a fluorescent hydrophobic probe such as 4, 4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS). The binding between bis-ANS and Sap C was pH-dependent, indicating that protonation leads to increased exposure of hydrophobic surfaces of Sap C. The interaction of Sap C with membranes, triggered by the development of hydrophobic properties at low pH values, was affected by the content of anionic phospholipids, such as phosphatidylserine or phosphatidylinositol, suggesting that anionic phospholipids have the potential to modulate the insertion of Sap C in the hydrophobic environment of lysosomal membranes. We previously showed that Sap C and anionic phospholipids are both required for the binding of glucosylceramidase to large vesicles. We have presently observed that Sap C is able to promote the association of glucosylceramidase with the lipid surface only when anionic phospholipids exceed a concentration of 5-10%. This level can be reached by summing lower amounts of individual anionic phospholipids, since they have additive effects. The present data extend and refine our model of the mechanism of glucosylceramidase activation and stress the key role of pH, Sap C and anionic phospholipids in promoting the interaction of the enzyme with membranes.


Asunto(s)
Glucosilceramidasa/química , Glicoproteínas/química , Fosfolípidos/química , Naftalenosulfonatos de Anilina , Animales , Aniones , Bovinos , Yema de Huevo , Colorantes Fluorescentes , Humanos , Concentración de Iones de Hidrógeno , Membranas Intracelulares/química , Lisosomas/química , Saposinas , Espectrometría de Fluorescencia , Proteínas Activadoras de Esfingolípidos
8.
FEBS Lett ; 336(1): 159-62, 1993 Dec 20.
Artículo en Inglés | MEDLINE | ID: mdl-8262201

RESUMEN

The function of saposin C (Sap C), a glucosylceramidase activator protein, in the enzyme stimulation by phosphatidylserine (PS) liposomes has been investigated. Using gel filtration experiments evidence was obtained for Sap C binding to PS large unilamellar vesicles (LUV) but not to glucosylceramidase. PS LUV, which by themselves are unable to tightly bind and stimulate the enzyme, acquire the capacity to also bind the enzyme after interaction with Sap C, making it express its full activity. Our results indicate that the primary step in the Sap C mode of action resides in its association with PS membranes; in turn, this association promotes the interaction between the membranes and glucosylceramidase.


Asunto(s)
Glucosilceramidasa/metabolismo , Glicoproteínas/fisiología , Fosfatidilserinas/metabolismo , Activación Enzimática , Humanos , Liposomas , Saposinas
9.
FEBS Lett ; 349(2): 181-6, 1994 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-8050562

RESUMEN

We have previously shown that saposin C (Sap C), a glucosylceramidase activator protein, interacts with phosphatidylserine (PS) large unilamellar vesicles (LUV), promoting the glucosylceramidase binding to the bilayer [(1993) FEBS Lett. 336, 159-162]. In the present paper the consequences of the Sap C interaction on the lipid organization of the vesicles are reported. It was found that Sap C perturbs the PS bilayer as shown by the release of an encapsulated fluorescent dye. Three different procedures, resonance energy transfer, gel filtration and electron microscopy, indicated that the activator protein is also able to make PS liposomes fuse. The effects of Sap C on PS vesicles were observed at low but not at neutral pH. The lipid composition of the bilayer also affected the Sap C-induced destabilization; in fact, the presence of PS in mixed LUV was essential for significant leakage to occur. These results demonstrate for the first time that Sap C is a protein capable of destabilizing and fusing acidic phospholipid-containing membranes in a pH-dependent fashion.


Asunto(s)
Glicoproteínas/metabolismo , Membrana Dobles de Lípidos/metabolismo , Fosfatidilserinas/metabolismo , beta-Glucosidasa/metabolismo , Animales , Bovinos , Cromatografía en Gel , Activación Enzimática , Glicoproteínas/química , Membrana Dobles de Lípidos/química , Liposomas , Fluidez de la Membrana , Fusión de Membrana , Fosfatidilserinas/química , Saposinas , beta-Glucosidasa/química
10.
FEBS Lett ; 216(2): 190-4, 1987 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-3582671

RESUMEN

A protein activator of glucosylceramidase (EC 3.2.1.45) has been previously identified by us in human placenta [(1985) Biochim. Biophys. Acta 836, 157-166]. In the present paper we report that its function in vitro is to stimulate the binding of the enzyme to its substrate, glucosylceramide. After the purification step which frees the enzyme of most of its activator protein (octyl-Sepharose 4B chromatography), the capacity of glucosylceramidase to bind to the glucosylceramide micelles is dramatically decreased. The addition of the activator protein to the purified enzyme restores this binding.


Asunto(s)
Cerebrósidos/metabolismo , Glucosidasas/metabolismo , Glucosilceramidasa/metabolismo , Glucosilceramidas/metabolismo , Glicoproteínas , Placenta/enzimología , Proteínas/metabolismo , Centrifugación por Gradiente de Densidad , Activación Enzimática , Humanos , Micelas , Unión Proteica , Saposinas , Proteínas Activadoras de Esfingolípidos
11.
Neuromuscul Disord ; 12(4): 386-91, 2002 May.
Artículo en Inglés | MEDLINE | ID: mdl-12062257

RESUMEN

We describe three brothers suffering from Krabbe's disease with onset in the fifth decade. The proband showed a complete deficiency of leukocyte enzyme galactocerebrosidase and was found to be heterozygous for two previously described mutations: G > A809 and 502T/del consisting of a 30 kb deletion. In all three brothers the neurological examination showed features of asymmetrical peripheral neuropathy associated with pyramidal signs and the electrophysiological examination showed a generalized slowing of nerve conduction velocities. Two patients died at 59 and 61 years of age due to respiratory failure. Both the proband and his brother underwent a sural nerve biopsy. In the former the most striking finding was the presence of uniformly thin myelin sheaths without evidence of demyelination; a complete absence of fibers was found in the latter. Our findings confirm that peripheral neuropathy may be the presenting feature of late-onset Krabbe's disease. Hypomyelination rather than demyelination may represent the distinguishing pathological finding of this condition.


Asunto(s)
Leucodistrofia de Células Globoides/complicaciones , Vaina de Mielina/patología , Enfermedades del Sistema Nervioso Periférico/complicaciones , Adulto , Edad de Inicio , Biopsia , Salud de la Familia , Humanos , Masculino , Microscopía Electrónica , Persona de Mediana Edad , Vaina de Mielina/ultraestructura , Núcleo Familiar , Enfermedades del Sistema Nervioso Periférico/patología , Nervio Sural/patología , Nervio Sural/ultraestructura
12.
Clin Biochem ; 20(6): 429-33, 1987 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-3124976

RESUMEN

A new protein activator of glucosylceramidase has recently been found in human placenta. In the present work, it has been compared with a previously reported glucosylceramidase activator, the Gaucher factor. The two activators showed different properties. The Gaucher factor stimulated 100% the 4-methylumbelliferyl-beta-D-glucopyranoside hydrolysis while the placental factor inhibited it 50%. Furthermore, the placental factor neither decreased the Michaelis constant, Km, nor increased the degree of inactivation by conduritol-beta-epoxide as the Gaucher factor does. From these results it has been concluded that the two activators are different substances. The activating effect of the placental factor is specific for the hydrolysis of glucosylceramide; neither the hydrolysis of glucosylsphingosine nor that of the 4-methylumbelliferyl derivative are enhanced by this protein. Owing to its specificity and high level in a human tissue, the placental factor is likely to have a physiological role in the catabolism of glucosylceramide.


Asunto(s)
Glucosidasas/metabolismo , Glucosilceramidasa/metabolismo , Placenta/fisiología , Extractos de Tejidos/farmacología , beta-Glucosidasa/metabolismo , Enfermedad de Gaucher/fisiopatología , Glicoproteínas/fisiología , Humanos , Cinética , Lisosomas/enzimología , Mitocondrias/enzimología , Placenta/enzimología , Saposinas , Bazo/fisiopatología , Especificidad por Sustrato
13.
Clin Chim Acta ; 131(1-2): 1-13, 1983 Jun 30.
Artículo en Inglés | MEDLINE | ID: mdl-6883703

RESUMEN

Our earlier observation that N-stearoyl- and N-lignoceroyl-glucosyl-dihydro-sphingosines have much lower affinity to the hydrolytic enzyme, glucosylceramidase, than the natural mixture of glucosylceramide [11] has been further pursued with catalytically hydrogenated natural substrate. Similar experiments were also carried out for hydrolysis of galactosylceramide of different structures by galactosylceramidase. The hydrogenation procedure completely saturated both fatty acid and long chain base moieties. For either enzyme, the hydrogenated natural substrate had affinity approximately half of the untreated natural substrate mixture. However, the synthetic glucosylceramides which contained a single saturated fatty acid and dihydrosphingosine had generally still lower affinity than the hydrogenated natural mixture. When two synthetic substrates of different fatty acids were mixed together, the affinity to the enzyme increased to a level much higher than that of either of the synthetic substrates alone and reached that of the hydrogenated natural substrate mixture. The findings were similar for galactosylceramide hydrolysis except that the synthetic substrate with palmitic of stearic acid had affinity to the enzyme not much lower than that of the hydrogenated natural substrate mixture. These effects of different structures on their enzymatic hydrolysis remained similar when other constituents of the assay mixture, such as the buffer and detergents, were varied.


Asunto(s)
Cerebrósidos/metabolismo , Galactosilceramidas/metabolismo , Glucosilceramidas/metabolismo , Cromatografía en Capa Delgada , Galactosilceramidasa/metabolismo , Glucosilceramidasa/metabolismo , Humanos , Hidrólisis , Cinética , Relación Estructura-Actividad
14.
Clin Chim Acta ; 118(1): 1-7, 1982 Jan 05.
Artículo en Inglés | MEDLINE | ID: mdl-7053901

RESUMEN

Commercially available [3H]glucosylceramide is derived from spleen tissue of patients with Gaucher's disease. When such tritiated glucosylceramide was diluted with unlabelled glucosylceramide from different sources and used as the substrate for assays of glucosylceramidase, the apparent activities obtained differed drastically. When diluted with synthetic N-stearoyl- or N-lignoceroyl-glucosyldihydrosphingosine, the release of [3H]glucose was 4-5 times greater than when diluted with unlabelled glucosylceramide from Gaucher spleen, with either brain or fibroblast homogenate as the enzyme source. The Ki values of the synthetic substrates were 15-30 times greater than the Km for the natural mixture, indicating much lower affinity of the synthetic substrates for the enzyme. Although the reason for the reduction in affinity could not be identified, caution is required when the commercial [3H]glucosylceramide is to be diluted with unlabelled glucosylceramide.


Asunto(s)
Cerebrósidos/metabolismo , Glucosidasas/metabolismo , Glucosilceramidasa/metabolismo , Glucosilceramidas/metabolismo , Encéfalo/enzimología , Fibroblastos/enzimología , Enfermedad de Gaucher/enzimología , Glucosa/metabolismo , Humanos , Cinética , Bazo/análisis
15.
Clin Chim Acta ; 172(2-3): 323-34, 1988 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-3370844

RESUMEN

Glucosylceramidase (EC 3.2.1.45) protein activators, similar to the 'placental factor' previously identified by us in human placenta, have also been found in human liver, normal and Gaucher fibroblasts and Gaucher spleen. They stimulate enzymatic hydrolysis of the natural substrate, glucosylceramide, but not that of the artificial substrate, 4-MU-beta-D-glucopyranoside. They are present in the tissues over the minimum amount necessary for full activation of the enzyme and must be eliminated from crude enzyme preparations in order to observer their effect on glucosylceramidase activity. The factors are not tissue-specific in that the factors from any one of the sources can activate glucosylceramidase from either placenta or liver. The presence of taurocholate or phosphatidylserine in the assay is essential for the factor efficiency. A normal level of the activator proteins was found in fibroblasts from subjects affected with Gaucher disease type I, type II and type III.


Asunto(s)
Glucosidasas/metabolismo , Glucosilceramidasa/metabolismo , Células Cultivadas , Activación Enzimática , Femenino , Fibroblastos/enzimología , Enfermedad de Gaucher/enzimología , Glucosilceramidas/metabolismo , Humanos , Hígado/enzimología , Placenta/enzimología , Embarazo , Bazo/enzimología
16.
Clin Genet ; 72(6): 538-42, 2007 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-17919309

RESUMEN

Gaucher disease is generally caused by a deficiency of the lysosomal enzyme glucocerebrosidase. The degradation of glycosphingolipids requires also the participation of sphingolipid activator proteins. The prosaposin PSAP gene codes for a single protein which undergoes post-translational cleavage to yield four proteins named saposins A, B, C and D. Saposin (SAP-) C is required for glucosylceramide degradation, and its deficiency results in a variant form of Gaucher disease. In this report, we present clinical, biochemical, and molecular findings in a 36-year-old man and his 30-year-old sister with non-neuronopathic Gaucher disease due to SAP-C deficiency. Very high levels of chitotriosidase activity, chemokine CCL18, and increased concentration of glucosylceramide in plasma and normal beta-glucosidase activity in skin fibroblasts were observed in the patients. A molecular genetics study of the PSAP gene enabled the identification of one missense mutation, p.L349P, located in the SAP-C domain and another mutation, p.M1L, located in the initiation codon of the prosaposin precursor protein. The presented findings describe the first cases where the non-neuronopathic Gaucher disease has been definitely demonstrated to be a consequence of SAP-C deficiency. Three previously described cases in the literature displayed a Gaucher type 3 phenotype.


Asunto(s)
Enfermedad de Gaucher/genética , Enfermedad de Gaucher/metabolismo , Saposinas/deficiencia , Saposinas/genética , Adulto , Femenino , Enfermedad de Gaucher/diagnóstico , Humanos , Masculino , Mutación Missense , Fenotipo
17.
Enzyme ; 42(2): 87-97, 1989.
Artículo en Inglés | MEDLINE | ID: mdl-2591351

RESUMEN

This paper reports the results of ultracentrifugation experiments devised for investigating the interactions occurring in the conditions of the enzymatic assay between glucosylceramidase and the components of the substrate dispersion. This dispersion contains, besides glucosylceramide, taurocholate and oleic acid. It has been found that glucosylceramide aggregates with oleic acid, while taurocholate is unable to associate with the sphingolipid, but improves the stability of the dispersion. When a crude glucosylceramidase placental preparation is incubated with the assay mixture the enzyme is almost totally bound to the glucosylceramide-oleic acid particles. The binding between glucosylceramidase and the substrate-containing particles is dramatically depressed by changes of experimental conditions which negatively influence also the enzyme activity such as: (1) a decrease in the molarity of the citrate/phosphate buffer; (2) an increase of the buffer pH, and (3) an increase of the taurocholate concentration. An excess of oleic acid neither inhibits the binding nor the activity. These results strongly suggest that glucosylceramidase activity is directly correlated with the binding of the enzyme to the lipid interface of the substrate-containing particles. We conclude that the enzymatic mechanism of glucosylceramide hydrolysis involves at least two steps: first the physical localization of the enzyme at the lipid-water interface, second the hydrolysis of the substrate glucosidic bond.


Asunto(s)
Glucosidasas/metabolismo , Glucosilceramidasa/metabolismo , Sitios de Unión , Centrifugación por Gradiente de Densidad , Enfermedad de Gaucher/enzimología , Glucosilceramidasa/aislamiento & purificación , Glicósido Hidrolasas/metabolismo , Humanos , Cinética , Lisosomas/enzimología , Mitocondrias/enzimología , Unión Proteica , Bazo/enzimología
18.
Enzyme ; 37(3): 115-26, 1987.
Artículo en Inglés | MEDLINE | ID: mdl-2953589

RESUMEN

Arylsulfatase C (ASC) was purified about 1,000-fold from human placenta. The major steps in the procedure included chromatography on Con A-Sepharose and Bio-Gel A-1.5 m. The purified enzyme was homogeneous by sodium dodecylsulfate/polyacrylamide gel electrophoresis. The native enzyme has an apparent molecular weight of 238,000 resulting from three identical subunits of 78,000 daltons. The purified enzyme hydrolyzes the artificial substrate p-nitrophenyl sulfate (NPS), and the two natural substrates estronesulfate (ES) and dehydroepiandrosterone sulfate (DHEAS), the ratio of these three activities being constant throughout the purification. ES and DHEAS are powerful competitive inhibitors of the enzymatic hydrolysis of NPS. ASC, ESase and DHEASase activities show the same thermal stability. These results strongly suggest that a single enzyme is responsible for the hydrolysis of the two natural and the artificial substrates.


Asunto(s)
Arilsulfatasas/aislamiento & purificación , Placenta/enzimología , Sulfatasas/aislamiento & purificación , Cromatografía , Deshidroepiandrosterona/análogos & derivados , Sulfato de Deshidroepiandrosterona , Estrona/análogos & derivados , Femenino , Humanos , Peso Molecular , Nitrobencenos , Embarazo , Conformación Proteica , Esteril-Sulfatasa , Especificidad por Sustrato
19.
Eur J Biochem ; 146(2): 315-21, 1985 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-3967661

RESUMEN

Properties of glucosylsphingosine (gluco-psychosine) glucosyl hydrolase were studied in detail in cultured human fibroblasts and placenta and were compared with those of glucosylceramidase. The two activities, that are deficient in tissues of Gaucher patients, showed minor but consistent differences. The pH optima were 4.8 for psychosine hydrolysis and 5.3 for glucosylceramide hydrolysis. In the presence of oleic acid, taurocholate activated glucosylceramidase more than 10-fold, while it activated psychosine hydrolysis only by about 30%. Triton X-100 was stimulatory for glucosylceramidase but was strongly inhibitory for psychosine hydrolysis. Phospholipids, that increase many times glucosylceramidase activity, were moderately inhibitory to enzymatic hydrolysis of psychosine. The psychosine hydrolase activity was slightly more heat-stable than the glucosylceramidase activity. The Km values for the two substrates were similar; 1.7 X 10(-5) M for psychosine and 2.7 X 10(-5) M for glucosylceramide. The V for glucosylceramide was, however, 100-times that for psychosine hydrolysis. Psychosine acted as a potent non-competitive inhibitor (Ki = 1.8 X 10(-5) M), while glucosylceramide was a weak inhibitor against psychosine hydrolysis. Within the limit of glucosylceramide solubility, psychosine hydrolysis could not be inhibited by more than 50%. Furthermore, the Dixon plot of glucosylceramide inhibition showed an anomalous slope. The ratio of the two activities was similar in fibroblasts, in the placenta mitochondria-lysosomal fraction and in a partially purified placental preparation. These findings are best explained by the hypothesis that, although the two substrates are hydrolyzed by a single enzyme, they share an overlapping but not identical catalytic site while binding to hydrophobic sites unique for the respective substrates.


Asunto(s)
Fibroblastos/enzimología , Glucosidasas/aislamiento & purificación , Glucosilceramidasa/aislamiento & purificación , Placenta/enzimología , Sitios de Unión , Fenómenos Químicos , Química , Femenino , Humanos , Hidrólisis , Piel/enzimología , Especificidad por Sustrato
20.
Anthropol Anz ; 42(4): 307-14, 1984 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-6099092

RESUMEN

Four Albanian and three Croatian communities settled in Molise (Italy) have been investigated for galactose-1-phosphate uridyltransferase (GALT) polymorphism. To obtain a detailed identification of each phenotype, electrophoresis and quantitative enzymatic analysis were performed on all samples. In addition to this isoelectric focusing was utilized to confirm and the D and LA variants. The gene frequencies of the different GALT alleles turned out to be: N = 0.912 (Albanians) and N = 0.868 (Croatians); G = 0.004 (Albanians); D = 0.051 (Albanians) and D = 0.081 (Croatians); LA = 0.033 (Albanians) and LA = 0.051 (Croatians). Both variants show frequencies similar to that observed in other Caucasoid populations.


Asunto(s)
Etnicidad , Nucleotidiltransferasas/genética , Polimorfismo Genético , UTP-Hexosa-1-Fosfato Uridililtransferasa/genética , Adolescente , Niño , Eritrocitos/enzimología , Femenino , Frecuencia de los Genes , Genotipo , Humanos , Italia , Masculino
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