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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.

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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.

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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.

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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.

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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.

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