Biogenic habitat shifts under long-term ocean acidification show nonlinear community responses and unbalanced functions of associated invertebrates.

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.

Avaliação do atendimento pelo programa Aprendendo com Saúde na Coordenadoria Regional de Saúde Sul da Secretaria Municipal de Saúde de São Paulo

O Programa Aprendendo com Saúde (APD) têm como objetivo a promoção, prevenção e a assistência à saúde do escolar, sendo normatizado em setembro de 2007 com o objetivo de ampliar e aperfeiçoar o Programa Municipal de Atenção á Saúde do Escolar

Avaliação do atendimento pelo programa Aprendendo com Saúde na Coordenadoria Regional de Saúde Sul da Secretaria Municipal de Saúde de São Paulo / Evaluation of the program by learning from the Health Regional Health South Municipal Health Secretariat of Sao Paulo

O Programa Aprendendo com Saúde (APD) têm como objetivo a promoção, prevenção e a assistência à saúde do escolar, sendo normatizado em setembro de 2007 com o objetivo de ampliar e aperfeiçoar o Programa Municipal de Atenção á Saúde do Escolar(AU)

Avaliação do atendimento pelo programa aprendendo com saúde na coordenadoria regional de saúde sul da secretaria municipal de saúde de São Paulo / Evaluation of the program by learning from the health regional health south municipal health secretariat of Sao Paulo

O Programa Aprendendo com Saúde (APD) têm como objetivo a promoção, prevenção e a assistência à saúde do escolar, sendo normatizado em setembro de 2007 com o objetivo de ampliar e aperfeiçoar o Programa Municipal de Atenção á Saúde do Escolar.

Non-neuronopathic Gaucher disease due to saposin C deficiency.

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.

Peripheral neuropathy with hypomyelinating features in adult-onset Krabbe's disease.

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.

Saposin D solubilizes anionic phospholipid-containing membranes.

Saposin (Sap) D is a late endosomal/lysosomal small protein, generated together with three other similar proteins, Sap A, B, and C, from the common precursor, prosaposin. Although the functions of saposins such as Sap B and C are well known (Sap B promotes the hydrolysis of sulfatides and Sap C that of glucosylceramide), neither the physiological function nor the mechanism of action of Sap D are yet fully understood. We previously found that a dramatic increase of Sap D superficial hydrophobicity, occurring at the low pH values characteristic of the late endosomal/lysosomal environment, triggers the interaction of the saposin with anionic phospholipid-containing vesicles. We have presently found that, upon lipid binding, Sap D solubilizes the membranes, as shown by the clearance of the vesicles turbidity. The results of gel filtration, density gradient centrifugation, and negative staining electron microscopy demonstrate that this effect is due to the transformation of large vesicles to smaller particles. The solubilizing effect of Sap D is highly dependent on pH, the lipid/saposin ratio, and the presence of anionic phospholipids; small variations in each of these conditions markedly influences the activity of Sap D. The present study documents the interaction of Sap D with membranes as a complex process. Anionic phospholipids attract Sap D from the medium; when the concentration of the saposin on the lipid surface reaches a critical value, the membrane breaks down into recombinant small particles enriched in anionic phospholipids. Our results suggest that the role played by Sap D is more general than promoting sphingolipid degradation, e.g. the saposin might also be a key mediator of the solubilization of intralysosomal/late endosomal anionic phospholipid-containing membranes.

Further studies on the reconstitution of glucosylceramidase activity by Sap C and anionic phospholipids.

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.

Structural and membrane-binding properties of saposin D.

Saposin D is generated together with three similar proteins, saposins A, B and C, from a common precursor, called prosaposin, in acidic organelles such as late endosomes and lysosomes. Although saposin D has been reported to stimulate the enzymatic hydrolysis of sphingomyelin and ceramide, its physiological role has not yet been clearly established. In the present study we examined structural and membrane-binding properties of saposin D. At acidic pH, saposin D showed a great affinity for phospholipid membranes containing an anionic phospholipid such as phosphatidylserine or phosphatidic acid. The binding of saposin D caused destabilization of the lipid surface and, conversely, the association with the membrane markedly affected the fluorescence properties of saposin D. The presence of phosphatidylserine-containing vesicles greatly enhanced the intrinsic tyrosine fluorescence of saposin D, which contains tyrosines but not tryptophan residues. The structural properties of saposin D were investigated in detail using advanced MS analysis. It was found that the main form of saposin D consists of 80 amino acid residues and that the six cysteine residues are linked in the following order: Cys5-Cys78, Cys8-Cys72 and Cys36-Cys47. The disulfide pattern of saposin D is identical with that previously established for two other saposins, B and C, which also exhibit a strong affinity for lipids. The common disulfide structure probably has an important role in the interaction of these proteins with membranes. The analysis of the sugar moiety of saposin D revealed that the single N-glycosylation site present in the molecule is mainly modified by high-mannose-type structures varying from two to six hexose residues. Deglycosylation had no effect on the interaction of saposin D with phospholipid membranes, indicating that the glycosylation site is not related to the lipid-binding site. The association of saposin D with membranes was highly dependent on the composition of the bilayer. Neither ceramide nor sphingomyelin, sphingolipids whose hydrolysis is favoured by saposin D, promoted its binding, while the presence of an acidic phospholipid such as phosphatidylserine or phosphatidic acid greatly favoured the interaction of saposin D with vesicles at low pH. These results suggest that, in the acidic organelles where saposins are localized, anionic phospholipids may be determinants of the saposin D topology and, conversely, saposin D may affect the lipid organization of anionic phospholipid-containing membranes.

Saposins and their interaction with lipids.

The lysosomal degradation of several sphingolipids requires the presence of four small glycoproteins called saposins, generated by proteolytic processing of a common precursor, prosaposin. Saposins share several structural properties, including six similarly located cysteines forming three disulfide bridges with the same cysteine pairings. Recently it has been noted that also other proteins have the same polypeptide motif characterized by the similar location of six cysteines. These saposin-like (SAPLIP) proteins are surfactant protein B (SP-B), 'Entamoeba histolytica' pore-forming peptide, NK-lysin, acid sphingomyelinase and acyloxyacyl hydrolase. The structural homology and the conserved disulfide bridges suggest for all SAPLIPs a common fold, called 'saposin fold'. Up to now a precise fold, comprising five alpha-helices, has been established only for NK-lysin. Despite their similar structure each saposin promotes the degradation of specific sphingolipids in lysosomes, e.g. Sap B that of sulfatides and Sap C that of glucosylceramides. The different activities of the saposins must reside within the module of the alpha-helices and/or in additional specific regions of the molecule. It has been reported that saposins bind to lysosomal hydrolases and to several sphingolipids. Their structural and functional properties have been extensively reviewed and hypotheses regarding their molecular mechanisms of action have been proposed. Recent work of our group has evidenced a novel property of saposins: some of them undergo an acid-induced change in hydrophobicity that triggers their binding to phospholipid membranes. In this article we shortly review recent findings on the structure of saposins and on their interactions with lipids, with special attention to interactions with phospholipids. These findings offer a new approach for understanding the physiological role of saposins in lysosomes.

Importance of splicing for prosaposin sorting.

The prosaposin gene encodes a 70 kDa protein. This protein might either reach the lysosomes and get processed there to four peptides, which are activators of known lysosomal enzymes, or be secreted by cells as a 70 kDa protein, recently anticipated to have several biological activities. The human prosaposin gene has a 9 bp exon (exon 8) that is alternatively spliced, thus encoding three prosaposin forms: one with an extra three amino acid residues, one with an extra two residues and a third form with no extra residues. With the aim of testing whether there is an association between the alternative splicing and the differential sorting of prosaposins, we cloned two human prosaposin cDNA forms in a T7/EMC/vaccinia virus-derived vector and expressed them in human cells. The results indicated that the prosaposin containing the three extra residues accumulated faster and in greater amounts in the medium, whereas the prosaposin with no extra residues was mainly destined for lysosomes. Point mutations created by mutagenesis in vitro in the 9 bp stretch had a diverse effect on prosaposin secretion. When supplied to cells in the medium, both prosaposins were endocytosed and reached the lysosomes, where they were processed to active saposin B and saposin C. The activities of the saposins were monitored qualitatively and quantitatively. Quantitatively, lipids were extracted from the cells, separated on TLC and measured fluorimetrically. Qualitatively, cells were detected by fluorescence microscopy.

Effect of saposins A and C on the enzymatic hydrolysis of liposomal glucosylceramide.

The degradation of glucosylceramide in lysosomes is accomplished by glucosylceramidase with the assistance of, at least, another protein, saposin C (Sap C), which is generated from a large precursor together with three other similar proteins, saposins A, B, and D. In the present study, we have examined the effects of saposins on the enzymatic hydrolysis of glucosylceramide inserted in large and small phospholipid liposomes. The glucosylceramide contained in large unilamellar vesicles (LUV) was degraded by glucosylceramidase at a rate 7-8-fold lower than glucosylceramide inserted in small unilamellar vesicles (SUV). The separate addition of either Sap A or Sap C to the LUV system partially stimulated the sphingolipid degradation while saposins B and D had no effect. In the presence of both Sap A and Sap C, the rate of sphingolipid degradation was higher than the sum of the rates with the two saposins individually, indicating synergism in their actions. The stimulatory effect of the two saposins depended on the incorporation of an acidic phospholipid such as phosphatidylserine (PS) into LUV. The characteristics of glucosylceramidase activation by Sap C were different from those of Sap A. Sap C increased the rate of hydrolysis of both the artificial water soluble substrate, 4-methylumbelliferyl-beta-D-glucopyranoside, and the lipid substrate, glucosylceramide, while Sap A only stimulated degradation of the sphingolipid. Also the binding properties of Saps A and C were markedly different. At acidic pH values, Sap C bound to PS-containing LUV and promoted the association of glucosylceramidase with the membrane. In contrast, Sap A had poor affinity for the membrane even in the presence of glucosylceramide; moreover, Sap A did not potentiate the capacity of Sap C to mediate glucosylceramidase binding. In conclusion, our results show that both Sap A and Sap C are required for maximal hydrolysis of glucosylceramide inserted in PS-containing LUV, that their effects are synergistic, and that their mode of action is different. Sap C is responsible for the membrane binding of glucosylceramidase, while Sap A stimulation is possibly related to its effect on the conformation of the enzyme. It can be envisaged that Sap A in conjunction with Sap C might have a physiological role in glucosylceramide degradation.

pH-dependent conformational properties of saposins and their interactions with phospholipid membranes.

Saposins A, B, C, and D are small lysosomal glycoproteins released by proteolysis from a single precursor polypeptide, prosaposin. We have presently investigated the conformational states of saposins and their interaction with membranes at acidic pH values similar to those present in lysosomes. With the use of phase partitioning in Triton X-114, experimental evidence was provided that, upon acidification, saposins (Sap) A, C, and D acquire hydrophobic properties, while the hydrophilicity of Sap B is apparently unchanged. The pH-dependent exposure of hydrophobic domains of Sap C and D paralleled their pH-dependent binding to large unilamellar vesicles composed of phosphatidylcholine, phosphatidylserine, and cholesterol. In contrast, the binding of Sap A to the vesicles was very restricted, in spite of its increased hydrophobicity at low pH. A low affinity for the vesicles was also shown by Sap B, a finding consistent with its apparent hydrophilicity both at neutral and acidic pH. At the acidic pH values needed for binding, Sap C and D powerfully destabilized the phospholipid membranes, while Sap A and B minimally affected the bilayer integrity. In the absence of the acidic phospholipid phosphatidylserine, the induced destabilization markedly decreased. Of the four saposins, only Sap C was able to promote the binding of glucosylceramidase to phosphatidylserine-containing membranes. This result is consistent with the notion that Sap C is specifically required by glucosylceramidase to exert its activity. Our finding that an acidic environment induces an increased hydrophobicity in Sap A, C, and D, making the last two saposins able to interact and perturb phospholipid membranes, suggests that this mechanism might be relevant to the mode of action of saposins in lysosomes.

Structural analysis of saposin C and B. Complete localization of disulfide bridges.

Saposins A, B, C, and D are a group of homologous glycoproteins derived from a single precursor, prosaposin, and apparently involved in the stimulation of the enzymatic degradation of sphingolipids in lysosomes. All saposins have six cysteine residues at similar positions. In the present study we have investigated the disulfide structure of saposins B and C using advanced mass spectrometric procedures. Electrospray analysis showed that deglycosylated saposins B and C are mainly present as 79- and 80-residue monomeric polypeptides, respectively. Fast atom bombardment mass analysis of peptide mixtures obtained by a combination of chemical and enzymatic cleavages demonstrated that the pairings of the three disulfide bridges present in each saposin are Cys4-Cys77, Cys7-Cys71, Cys36-Cys47 for saposin B and Cys5-Cys78, Cys8-Cys72, Cys36-Cys47 for saposin C. We have recently shown that saposin C interacts with phosphatidylserine-containing vesicles inducing destabilization of the lipid surface (Vaccaro, A. M., Tatti, M., Ciaffoni, F., Salvioli, R., Serafino, A., and Barca, A. (1994) FEBS Lett. 349, 181-186); this perturbation promotes the binding of the lysosomal enzyme glucosylceramidase to the vesicles and the reconstitution of its activity. It was presently found that the effects of saposin C on phosphatidylserine liposomes and on glucosylceramidase activity are markedly reduced when the three disulfide bonds are irreversibly disrupted. These results stress the importance of the disulfide structure for the functional properties of the saposin.

Saposin C induces pH-dependent destabilization and fusion of phosphatidylserine-containing vesicles.

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.

Function of saposin C in the reconstitution of glucosylceramidase by phosphatidylserine liposomes.

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.

Studies on glucosylceramidase binding to phosphatidylserine liposomes: the role of bilayer curvature.

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.

Reconstitution of glucosylceramidase on binding to acidic phospholipid-containing vesicles.

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.

Effect of experimental conditions on the appearance of distinct forms of placental glucosylceramidase: use of gel filtration analysis as a means of ascertaining their occurrence.

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.

Correlation between the activity of glucosylceramidase and its binding to glucosylceramide-containing liposomes. Role of acidic phospholipids and fatty acids.

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.