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Hydrolysis of GM1-ganglioside by human liver beta-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.

SUBMITTER: Ho MW

PROVIDER: S-EPMC1165961 | biostudies-other | 1973 Oct

REPOSITORIES: biostudies-other

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Project description:Lysosomal beta-galactosidase (beta-Gal) occurs either alone in monomeric and dimeric forms, or in a high-M(r) complex with at least two additional proteins. One is neuraminidase and the second is the protective protein, which has also been shown to possess carboxypeptidase activity. beta-Gal activity is deficient in GM1-gangliosidosis as a primary defect, and is secondarily affected in galactosialidosis (GS), where the primary defect is the absence of protective protein activity. Fibroblasts from three patients with GM1-gangliosidosis, type 1, showed markedly reduced amounts of beta-Gal cross-reacting material (CRM), and a fourth appeared to have normal levels. A patient with type 2 GM1-gangliosidosis was also found to be CRM-normal. These findings demonstrate that patients with GM1-gangliosidosis type 1 are heterogeneous with respect to the level of residual beta-Gal protein. Fibroblasts from four patients with GS were strongly CRM-positive with an anti-beta-Gal antibody, as was a sample of brain from one of these patients, suggesting that the loss of beta-Gal activity is linked to a subtler change in the primary structure of the enzyme than has been previously thought. While three GS cell lines displayed reduced carboxypeptidase activity (to 32-42% of the control), one cell line was completely devoid of activity, demonstrating that while carboxypeptidase activity is a property of the protective protein this action is distinct and separate from its protective role. On direct immunoprecipitation with anti-beta-Gal antibody, a portion of the total carboxypeptidase activity co-precipitated with beta-Gal from extracts of normal and GM1-gangliosidosis cells, consistent with the presence of the complex in these cells. However, no carboxypeptidase activity was precipitable with this antibody from GS fibroblasts, suggesting the absence of complex from these cells. To examine this further, the various forms of beta-Gal were resolved by h.p.l.c. molecular-sieve chromatography. Three forms of beta-Gal activity were resolved in normal cells: a complex, a dimer and a monomer. Residual beta-Gal activity of GS cells resolved into two of these forms, the complex and the monomer. In normal and GM1-gangliosidosis cells a portion of the total carboxypeptidase activity co-chromatographed with the complex while the bulk of the activity occurred in a single 36,000-M(r) peak. Only the low-M(r) carboxypeptidase activity was detected in GS cells. This confirms our results on immunoprecipitation indicating that portions of the beta-Gal and the carboxypeptidase activities exist outside the complex in normal, GM1-gangliosidosis and GS cells. In summary, the loss of protective protein function from GS cells results in disproportionate loss of the dimeric and monomeric forms of beta-Gal activity, but does not result in the complete degradation of the protein.

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Hydrolysis of GM1-ganglioside by human liver beta-galactosidase isoenzymes.

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