Human placental beta-galactosidase. Characterization of the dimer and complex forms of the enzyme.
GM1 ganglioside beta-galactosidase (beta-Gal) is deficient in the autosomal recessive disorder GM1 gangliosidosis. A portion of the enzyme occurs in a complex with neuraminidase and an additional glycoprotein, protective protein, but the nature of the interactions conferring the stability of the complex is unknown. Affinity chromatography of beta-Gal on p-aminophenylthiogalactose-Sepharose (PATG-Sepharose) at pH 4.3, the pH optimum of beta-Gal, resulted in a 260-fold enrichment of beta-Gal, but the major protein in the fraction had an M(r) value of 74,000. Affinity chromatography on PATG-Sepharose at pH 5.2 showed substantial enrichment (4000-fold) of beta-Gal, and the mature form of the enzyme (M(r) 64,000) was the major protein in the preparation. Using h.p.l.c. molecular-sieve chromatography, we found that about 15% of the total beta-Gal occurred in a high-M(r) form (greater than 600,000), the presumptive complex, with 85% eluting at M(r) 150,000, suggestive of a dimer. This distribution was independent of both high (60 mg/ml) and low (5 mg/ml) protein concentration and the pH (pH 4.3 or 5.2) of the sample applied to the column. Furthermore, incubation for 90 min at 37 degrees C, conditions which had previously been suggested as optimal for formation of the complex, had no effect on this distribution. Further fractionation by anion-exchange chromatography and a second affinity column step yielded a beta-Gal preparation that contained a single polypeptide chain (M(r) 64,000), was devoid of neuraminidase and protective protein (absent carboxypeptidase activity), and when injected into rabbits gave rise to monospecific rabbit antisera. We conclude that the protein composition of the complex is variable (i.e. it is different when isolated at pH 4.3 and 5.2) and that the amount of beta-Gal tightly associated with the complex constitutes a small fraction of the total beta-Gal activity. The more prevalent form of the enzyme is a beta-Gal homodimer that is stable and devoid of either neuraminidase activity or protective protein.
Hubbes
M
M
Research Institute, Hospital For Sick Children, Toronto, Ontario, Canada.
D'Agrosa
R M
RM
Callahan
J W
JW
D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D005260:Female; D006801:Humans; D006863:Hydrogen-Ion Concentration; D046911:Macromolecular Substances; D008970:Molecular Weight; D009439:Neuraminidase / Q000378:metabolism; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy; D001616:beta-Galactosidase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D046911:Macromolecular Substances; D009439:Neuraminidase; D001616:beta-Galactosidase