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1. Acid alpha-glucosidase was purified 3500-fold from rabbit muscle. 2. The enzyme was activated by cations, the degree of activation varying with the substrate. Enzyme action on glycogen was most strongly activated and activation was apparently of a non-competitive type. With rabbit liver glycogen as substrate, the relative V(max.) increased 15-fold, accompanied by an increase in K(m) from 8.3 to 68.6mm-chain end over the cation range 2-200mm-Na(+) at pH4.5. Action on maltose was only moderately activated (1.3-fold, non-competitively) and action on maltotriose was marginally and competitively inhibited. 3. The pH optimum at 2mm-Na(+) was 4.5 (maltose) and 5.1 (glycogen). Cation activation of enzyme action on glycogen was markedly pH-dependent. At 200mm-Na(+), the pH optimum was 4.8 and activity was maximally stimulated in the range pH4.5-3.3. 4. Glucosidase action on maltosaccharides was associated with pronounced substrate inhibition at concentrations exceeding 5mm. Of the maltosaccharides tested, the enzyme showed a preference for p-nitrophenyl alpha-maltoside (K(m) 1.2mm) and maltotriose (K(m) 1.8mm). The extrapolated K(m) for enzyme action on maltose was 3.7mm. 5. The macromolecular polysaccharide substrate glycogen differed from linear maltosaccharide substrates in the kinetics of its interaction with the enzyme. Activity was markedly dependent on pH, cation concentration and polysaccharide structure. There was no substrate inhibition. 6. The enzyme exhibited constitutive alpha-1,6-glucanohydrolase activity. The K(m) for panose was 20mm. 7. The enzyme catalysed the total conversion of glycogen into glucose. The hydrolysis of alpha-1,6-linkages was apparently rate-limiting during the hydrolysis of glycogen. 8. Enzyme action on glycogen and maltose released the alpha-anomer of d-glucose. 9. The results are discussed in terms of the physiological role of acid alpha-glucosidase in lysosomal glycogen catabolism.

1. The maltase and glucoamylase activities of acid alpha-glucosidase purified from rabbit muscle exhibited marked differences in certain physicochemical properties. These included pH stability, inactivation by thiol-group reagents, inhibition by alphaalpha-trehalose, methyl alpha-d-glucoside, sucrose, turanose, polyols, glucono-delta-lactone and monosaccharides, pH optimum and the kinetics and pH-dependence of cation activation. 2. The results are interpreted in terms of the existence of at least two specific substrate-binding sites or sub-sites. One site is specific for the binding of maltose and probably other oligosaccharides. The second site binds polysaccharides such as glycogen. 3. The sites appear to be in close proximity, since glycogen and maltose are mutually inhibitory substrates and interact directly in transglucosylation reactions. 4. Acid alpha-glucosidase exhibited intrinsic transglucosylase activity. The enzyme catalysed glucosyl-transfer reactions from [(14)C]maltose (donor substrate) to polysaccharides (glycogen and pullulan) and to maltose itself (disproportionation). The pH optimum was 5.1, with a shoulder or secondary activity peak at pH5.4. The glucose transferred to glycogen was attached by alpha-1,4- and alpha-1,6-linkages. Three major oligosaccharide products of enzyme action on maltose (disproportionation) were detected. 5. The kinetics of enzyme action on [(14)C]maltose showed that the rate of transglucosylation increased in a sigmoidal fashion as a function of substrate concentration, approximately in parallel with a decrease in the rate of glucose release. 6. The results are interpreted to imply competitive interaction at a specific binding site between maltose and water as glucosyl acceptors. 7. The results are discussed in terms of the possible existence of multiple subgroups of glycogen-storage disease type II.