Factors affecting the hydrolysis of ceramide-3 by alpha-galactosidase A from human liver

1. The effect of detergents on the catalytic properties of alpha-galactosidase from human liver was studied using p-nitrophenyl-alpha-galactoside and galactosyl-alpha(1 leads to 4)-galactosyl-beta(1 leads to 4)-glucosylceramide (ceramide-3) as substrates. 2. The hydrolysis of p-nitrophenyl-alpha-galactoside by alpha-galactosidase was inhibited by commercial preparations of sodium taurocholate and by taurocholate purified from these preparations by thin-layer chromatography. The extent of inhibition was dependent on the concentration of the detergent and on the amount of protein present. The impurities present in the preparation also inhibited the hydrolysis. 3. The inhibition of taurocholate preparations of p-nitrophenyl-alpha-galactoside hydrolysis was pH-dependent. 4. The inhibition by taurocholate of p-nitrophenyl-alpha-galactoside hydrolysis can be partly overcome by adding glycosphingolipids. 5. No significant hydrolysis of ceramide-3 occurs in the absence of detergent. Upon adding increasing concentrations of taurocholate, the rate of hydrolysis increases to a maximum value. At still higher taurocholate concentrations the activity decreases. 6. The concentrations of taurocholate giving a maximal rate of hydrolysis of ceramide-3 is dependent on the amount of protein present and independent of the ceramide-3 concentration. 7. When the pH dependence of the rate of hydrolysis of ceramide-3 was measured in the presence of a commercially available preparation of pure taurocholate or of crude taurocholate, curves with different shapes were obtained.