Further characterization of two forms of N-acetyl-α-galactosaminidase from human liver

Abstract

1. 1. 2 form of N-acetyl-α-galactosaminidase (EC 3.2.1.22) can be isolated from human liver using Sepharose 4B-concanavalin A chromatography, followed by CM-cellulose chromatography. Both forms hydrolyse not only p-nitrophenyl- N-acetyl-α-galactosaminide but also p-nitrophenyl-α-galactoside. These two forms were formerly known as α-galactosidase B and A-like α-galactosidase (Schram, A.W., Hamers, M.N., Brouwer-Kelder, B., Donker-Koopman, W.E. and Tager, J.M. (1977) Biochim. Biophys. Acta 482, 125–137; Schram, A.W., Hamers, M.N. and Tager, J.M. (1977) Biochim. Biophys. Acta 482, 138–144). 2. 2. In fresh normal liver, most of the N-acetyl-α-galactosaminidase activity is found in the fractions containing α-galactosidase B and a minor portion in the fractions containing α-galactosidase A. However, the minor peak of N-acetyl-α-galactosaminidase activity does not coincide with the peak of α-galactosidase A activity. Both peaks of N-acetyl-α-galactosaminidase activity can be precipitated by antiserum raised against α-galactosidase B but not by antiserum raised against α-galactosidase A. 3. 3. In fresh Fabry liver (which contains no α-galactosidase A) more than 98% of the N-acetyl-α-galactosaminidase activity was recovered in the fractions containing α-galactosidase B. Less than 2% was found in the fractions containing A-like α-galactosidase. Upon aging of the liver, the yield of the latter form increased (to almost 25% after 17 months storage at −20°C). 4. 4. The conversion of N-acetyl-α-galactosaminidase to the second form upon aging was accompanied by a decrease in apparent molecular weight from 110 000 ± 5000 to 99 000 ± 3000, as measured by Sephacryl S-200 chromatography. 5. 5. The kinetic properties of the enzyme change slightly upon aging. In contrast, there is no effect of aging on the pH optimum of the enzyme. 6. 6. This is concluded that only one lysosomal N-acetyl-α-galactosaminidase is present in human liver and that one of the two forms of N-acetyl-α-galactosaminidase observed in extracts of liver is formed by chemical modification of the enzyme during storage of the liver or during storage of the purified preparation.