Isolation and Characterization of Ceramide Trihexosidases (Form A) from Human Plasma

A procedure is presented for the purification from human plasma of two homogeneous and non-interconvertible forms of ceramide trihexosidase, designated Form A-1 and Form A-2. These enzymes exhibit the same electrophoretic characteristics on isoelectric focusing and 1% sodium dodecyl sulfate polyacrylamide gel electrophoresis. They have molecular weights of about 95,000, as determined by sucrose density gradient centrifugation and gel filtration, and consist of subunits with molecular weights of about 22,000. They are similar in heat stability, although Form A-2 retains 25% of its activity after boiling for 30 min, whereas Form A-1 is completely inactivated under the same conditions. Form A-1 is soluble in butanol and retains enzymatic activity in the organic solvent; Form A-2 retains enzymatic activity in 20% butanol but is not partitioned into the upper phase in a butanol-water system. Neither enzyme has any demonstrable activity with p-nitrophenyl-α-galactoside. The kinetic characteristics of Form A-1 ceramide trihexosidase can be summarized as follows. (a) In the absence of sodium taurocholate and sodium chloride, the enzyme has sigmoidal kinetics, whereas the kinetics are hyperbolic in the presence of 0.03 m sodium taurocholate and 0.15 m sodium chloride, with half-maximal velocity at a substrate concentration of 4.5 x 10-4m. (b) In the presence of optimal sodium taurocholate and sodium chloride, the enzyme is competitively inhibited by digalactosylceramide and the trisaccharide derived from the lipid substrate. (c) In the absence of sodium taurocholate and sodium chloride, digalactosyl ceramide is an activator at low substrate concentrations and an inhibitor at high substrate concentrations. (d) The enzyme is not inhibited by lactosylceramide, galactose, myo-inositol, p-nitrophenyl-α-galactoside, or 4-methylumbelliferyl-α-galactoside. (e) The enzyme becomes inactive when concentrated above 1.68 x 10-7m in an incubation. The kinetic characteristics of Form A-2 ceramide trihexosidase are as follows. (a) In the absence of sodium taurocholate and sodium chloride, the enzyme exhibits sigmoidal kinetics, whereas the kinetics are hyperbolic in the presence of 0.04 m sodium taurocholate and 0.15 m sodium chloride, with half-maximal velocity at a substrate concentration of 5 x 10-4m. (b) The enzyme is inhibited by the products of the reaction, galactose and lactosylceramide. (c) The enzyme is competitively inhibited by myo-inositol, digalactosylceramide, and t