Hydrolysis of GM1-ganglioside by human liver β-galactosidase isoenzymes

Abstract

1. GM(1)-ganglioside, specifically tritiated in the terminal galactose, was hydrolysed by two forms of ;acid' methylumbelliferyl beta-galactosidase isolated on gel filtration. 2. Identification of GM(1)-ganglioside beta-galactosidase activity with the ;acid' methyl-umbelliferyl beta-galactosidases was based on the following: coincident elution profiles on gel filtration; simultaneous inactivation by heat and other treatments; stabilization of both activities by chloride ions; mutual inhibition of hydrolysis by the two substrates. 3. The two isoenzymes (I) and (II) showed general requirements for a mixture of anionic and nonionic detergents in the hydrolysis of the natural substrate. 4. Isoenzyme (I) differed from (II) in molecular size, pH-activity profile, relative resistance to dilution and in sensitivity to various inhibitors. 5. The most significant difference between the isoenzymes is in substrate saturation kinetics: (I) was hyperbolic whereas (II) was sigmoid. The apparent Michaelis constants were 28mum for (I) and 77mum for (II). Isoenzyme (I) was insensitive to GM(2)-ganglioside whereas (II) was inhibited, consistent with the hypothesis that GM(1)-ganglioside (and its analogue) acts as modifier in isoenzyme (II) but not in (I). 6. Isoenzyme (I) was membrane-bound whereas (II) was soluble; the former probably represents isoenzyme (II) bound to membrane components, thereby becoming activated. 7. Membranes may serve a dual role in enzyme catalysis involving lipids: as a medium where both enzyme and substrate are effectively concentrated, and as actual activator of enzymes through binding of the latter to specific membrane components.

An electrophoretic variant of beta-galactosidase with altered catalytic properties in a patient with GM1 gangliosidosis.

“…In fact, the very high activity of this enzyme spuriously elevated the activity of "acid" 4-methylumbelliferyl j3-galactosidase (assayed at pH 4.3) to 17.9% of normal. We say "spuriously" since "acid" 4-methylumbelliferyl fl-galactosidase has been equated with GM1 0-galactosidase activity (4,5), and the correlation between these appears close provided neutral f-galactosidase is not elevated. The reason for the high activity of the neutral enzyme in C.E.…”

“…In liver and brain, most of the enzyme activity occurs in two forms termed "A" (65,000-75,000 molecular weight) and "B" (600,000-800,000 molecular weight) (4)(5)(6). In liver, a third form, "neutral" P-galactosidase, hydrolyzes low-molecular-weight synthetic P-galactosides and p-glucosodes but not GM, (4)(5)(6)(7). We have previously isolated very highly purified P-galactosidase A from human liver and raised antiserum against it; these antibodies crossreacted with P-galactosidase B but not with neutral p-galactosidase (8).…”

Separation of β-Galactosidases and β-Glucosidases from Human Liver

“…1.23)-active components of which one also possessed a ß-glucosidase (ß-Dglucoside glucohydrolase, EC 3.2.1.21) activity were demonstrated in the soluble fraction of normal human liver by gel chromatography and starchgel electrophoresis [3,4,6]. The two forms with the higher molecular weight have acidic pH optima (pH 4.0-4.4), are thermolabile, and are stimulated by chloride ions [3,4,6].…”

“…1.23)-active components of which one also possessed a ß-glucosidase (ß-Dglucoside glucohydrolase, EC 3.2.1.21) activity were demonstrated in the soluble fraction of normal human liver by gel chromatography and starchgel electrophoresis [3,4,6]. The two forms with the higher molecular weight have acidic pH optima (pH 4.0-4.4), are thermolabile, and are stimulated by chloride ions [3,4,6]. These two acid isoenzymes are sialoproteins which can be bound to concanavalin A insolubilized on agarose and eluted with methyl-a-D-mannoside [7], The major acid isoenzyme (ß-galactosidase A) has been purified and shown to possess enzymatic activity toward a group of synthetic and natural substrates [8], A structural relationship between the two acidic ß-galactosidases was suggested because of their similar substrate specificities, concomitant absence in inherited disease, and immunologic cross-reactivity [8].…”

The expression of the [Bgl] genetic complex during preimplantation mouse embryogenesis and in ovulated eggs

“…Many lysosomal enzyme de ficiency diseases result in storage dissorders of mucopolysaccharides, sphingolipids and glycolipids (Neufeld, Lim and Shapiro, 1975). In humans, the g-galactosidase enzyme with activity toward artificial substrates shows activity toward GM^ gangliosides (Ho, Cheetham and Robinson, 1973;Norden, Tennant and O'Brien, 1974). Deficiency diseases of this enzyme are characterized by an accumulation of glycopotein and GMp Enders and Schlafke (1974) detected a thin mucopolysaccharide coat on the embryo cell surface.…”