Purification and Properties of Human α-Galactosidases

Abstract

The thermolabile α-galactosidase (α-galactosidase A) and thermostable α-galactosidase (α-galactosidase B) were separated and purified from human placenta. A homogeneous α-galactosidase B preparation was obtained, but the α-galactosidase A preparation contained small amounts of contaminating protein and various other acid hydrolase activities. Each preparation had a molecular weight of approximately 150,000, as estimated by Sephadex filtration. α-Galactosidase A had a Km of 3.4 mm for the artificial substrate, 4-methylumbelliferyl-α-d-galactopyranoside, and of 40.6 mm for melibiose. α-Galactosidase B hydrolyzed 4-methylumbelliferyl-α-d-galactopyranoside with first order kinetics and appeared to have no activity with melibiose. Both enzymes had maximal enzyme activity at pH 4.5, but α-galactosidase A had a broad pH-activity curve, while that of the B enzyme was sharply peaked. α-Galactosidase A was inhibited by myoinositol; α-galactosidase B was not. The isoelectric point of α-galactosidase A was 4.70 ± 0.07; the isoelectric point of α-galactosidase B was 4.42 ± 0.04. Antibodies were produced against both the α-galactosidase A and α-galactosidase B preparations. No cross reactivity between the two enzyme preparations was found on double immunodiffusion. Neither antiserum neutralized enzyme activity, but the anti-α-galactosidase A serum precipitated α-galactosidase A activity from solution and the anti-α-galactosidase B serum precipitated α-galactosidase B activity from solution. Treatment of α-galactosidase A with neuraminidase does not change its immune reactivity or kinetic properties. These studies lend no support to the concept that α-galactosidase A is the neuraminyl derivative of galactosidase B or that the two enzymes are closely structurally related.