The enzymic hydrolysis of three synthetic sphingomyelins, spread as monomolecular films at the air/water interface by purified Staphylococcus aureus sphingomyelinase was studied. Each of the three sphinomyelins (DL-erythro-N-palmitoyl-, -N-stearoyl- and -N-lignoceryl-sphingosylphosphocholine) has an optimal activity-dependent surface pressure or concentration curve. The optimal surface pressure as well as the optimal surface density for hydrolysis was different for each of the three substrates. This optimum coincides with the liquid-condensed/liquid-expanded phase transition for each of the sphingomyelins. At initial surface pressures (pi 0) below the optimum, reaction rates are controlled mainly by surface density of the substrate; above the optimal pi 0, reaction rates decrease with increasing surface pressure. The difference between the three synthetic sphingomyelins are explained by the variation in the degree of asymmetry between their two paraffinic chains.

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Two sepharose-bound 1-deoxynojirimycin N-alkyl derivatives, N-(9-carboxynonyl)- and N-(l l-carboxyundecyl)-deoxynojirimycin, were used for the affinity purification of acid ß-glucosidase (ß-Glc) from normal and type-1 Ashkenazi Jewish Gaucher disease (AJGD) sources. The capacities of these nondegradable inhibitor supports were 0.5 and 0.75 mg of normal ß-Glc/ml of settled gel, respectively. The purified normal enzyme (14- 18% yield) had a specific activity of 1.6 X 10^6 nmol/h/mg protein and was homogeneous as evidenced by a single protein species of M(r) = 67,000 on sodium dodecylsulfate-polyacrylamide gel electrophoresis and reverse phase high-performance liquid chromatography (HPLC). Microsequencing demonstrated a single N terminus, and the sequence of the first 22 N-terminal amino acids was colinear with that predicted from the ß-Glc cDNA. Amino acid composition analyses of ß-Glc revealed a high content (35%) of hydrophobic amino acids. The N-decyl-deoxynojirimycin support facilitated the purification of the residual enzyme from type-1 AJGD spleen to about 7,500-fold in four steps with a yield of about 11 %. These new affinity supports provided improved stability, capacity and/or specificity compared to other affinity or HPLC methods for purifying this lysosomal glycosidase.

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