Purification and Properties of Human a-Galactosidases*

The thermolabile oc-galactosidase (cy-galactosidase A) and thermostable a-galactosidase (cr-galactosidase B) were separated and purified from human placenta. A homogeneous a-galactosidase B preparation was obtained, but the a-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. cY-Galactosidase A had a Km of 3.4 mM for the artil%cial substrate, 4-methylumbelliferyl-cr-n-galactopyranoside, and of 40.6 mu for melibiose. a-Galactosidase B hydrolyzed 4-methylumbelliferyl-cY-n-galactopyranoside with first order kinetics and appeared to have no activity with melibiose. Both enzymes had maximal enzyme activity at pH 4.5, but a-galactosidase A had a broad pa-activity curve, while that of the B enzyme was sharply peaked. cr-Galactosidase A was inhibited by myoinositol; cr-galactosidase B was not. The isoelectric point of a-galactosidase A was 4.70 =t 0.07; the isoelectric point of oc-galactosidase B was 4.42 f 0.04. Antibodies were produced against both the cY-galactosidase A and a+galactosidase B preparations. No cross reactivity between the two enzyme preparations was found on double immunodiffusion. Neither antiserum neutralized enzyme activity, but the anti-cr-galactosidase A serum precipitated oc-galactosidase A activity from solution and the anti-crgalactosidase B serum precipitated oc-galactosidase B activity from solution. Treatment of a-galactosidase A with neuraminidase does not change its immune reactivity or kinetic properties. These studies lend no support to the concept that cu-galactosidase A is the neuraminyl derivative of galactosidase B or that the two enzymes are closely structurally related.