New technique for enzymic hydrolysis of glycosphingolipids

Abstract

A method is described for the study of glycosyl ceramide glycosyl hydrolases. Problems arising from the limited solubility of glycosyl ceramides in aqueous media were overcome by coating the substrate on a filter paper disc that had been treated with phosphatidyl choline. A comparison between the disc method and conventional dispersion of the substrate by detergents was made with two enzymes, galactosylgalactosylglucosyl ceramide galactosyl hydrolase (trihexosyl ceramide galactosyl hydrolase) from lysosomes of human and rat small intestine and human spleen, and d-galactose oxidase. In both cases enzymatic activity was greater with the paper disc method than it was with substrates dispersed by detergents. The galactose liberated by the glycosyl hydrolase was determined as the trimethylsilyl derivative of the free sugar by gas-liquid chromatography.

Keywords

• glycosyl ceramide galactosyl hydrolase
• filter paper disc
• lipid dispersion
• d-galactose
• gas-liquid chromatography
• trimethylsilyl ether