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.
Vaccaro
A M
AM
Department of Metabolism and Pathological Biochemistry, Istituto Superiore Sanita', Roma, Italy.
Salvioli
R
R
Gallozzi
E
E
Ciaffoni
F
F
Tatti
M
M
D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D005260:Female; D005962:Glucosylceramidase / Q000032:analysis* / Q000378:metabolism; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008247:Lysosomes / Q000201:enzymology; D008861:Microsomes / Q000201:enzymology; D010920:Placenta / Q000201:enzymology* / Q000648:ultrastructure; D011247:Pregnancy
D006146:Guanidines; D005962:Glucosylceramidase; D019791:Guanidine