Purification and properties of the two major isozymes of alpha-galactosidase from human placenta

Kusiak, J.W.; Quirk, J.M.; Brady, R.O.

Journal of Biological Chemistry 253(1): 184-190

1978


ISSN/ISBN: 0021-9258
PMID: 201618
Accession: 068523854

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Abstract
Two .alpha.-galactosidase (.alpha.-D-galactoside galactohydrolase, EC 3.2.1.22) isozymes were highly purified from human placental tissue. Both isozymes appeared to be homogeneous based upon disc gel electrophoresis. Both were bound by concanavalin A-Sepharose and stained positively with the periodic acid-Schiff stain indicating that each contained carbohydrate. The A isozyme had a MW of 103,000 and the B isozyme 117,000 monitored by gel filtration and sodium dodecyl sulfate electrophoresis. Based upon sodium dodecyl sulfate electrophoresis under denaturing conditions, the A isozyme had a minimum subunit MW of 57,700 and the B, 47,700, indicating that each isozyme is a dimer of equal MW subunits. .alpha.-Galactosidase A had a Km of 1.55 mM for the artificial substrate 4-methylumbelliferyl-.alpha.-D-galactopyranoside while .alpha.-galactosidase B had a Km of 13.1 mM. Purified A isozyme was 300 times more active with the natural substrate, ceramide trihexoside (CTH, Gal-Gal-Glu-ceramide) than purified B isozyme. Each isozyme exhibited a different pattern of inhibition by various carbohydrates. .alpha.-Galactosidase A was heat-labile and susceptible to neuraminidase treatment. .alpha.-Galactosidase B was heat-stable, and not susceptible to neuraminidase treatment. Both isozymes had a pH optimum of 4.4 with the artificial substrate 4-methylumbelliferyl-.alpha.-D-galactopyranoside and the A isozyme had a pH optimum of 4.1 with ceramide trihexoside. .alpha.-Galactosidase A had a pI of 4.7 and the B isozyme had a pI of 4.4. Only artificial .alpha.-galactosidase and ceramide trihexosidase activities co-purified with the A isozyme while artificial .alpha.-galactosidase, ceramide trihexosidase and p-nitrophenol-2-acetamido-.alpha.-D-galactopyranosidase activities co-purified with the B isozyme. Albumin enhanced the artificial and natural activity of the A isozyme and the artificial activity of the B isozyme. These results support the theory that the 2 major isozymes of .alpha.-galactosidase from human placenta are not structurally related and do not share a precursor-product relationship.