Further purification and characterization of the acid α-glucosidase

Abstract

1. Centrifugation of rat liver acid glucosidase, which had been purified by adsorption on dextran gel, on a density gradient of sucrose showed the enzyme to be impure. 2. Preliminary purification of the enzyme before the gel filtration improved the final degree of purity of this preparation. Disc gel electrophoresis of this preparation showed a single band of protein. 3. The sedimentation co-efficient and the molecular weight determined on a sucrose gradient were 4.9-5.1s and 76000-83000 respectively for the rat liver enzyme, and 5.6s and 97000 for the acid alpha-glucosidase purified by means of the same procedure from the human kidney. 4. The Michaelis constants of rat liver and human kidney enzyme were 4.7x10(-3)m and 13.6x10(-3)m respectively with maltose as substrate. 5. The enzyme from both tissues was inhibited by tris and by erythritol. The inhibition of the rat liver acid glucosidase by erythritol was competitive.

Selective Determination of the Activities of Neutral and Acid α-Glucosidase Using Discontinuous Assays

“…Using data from figure 5, /C m -values for acid α-glucosidase were determined by Michaelis-Menten, Lineweaver-Burk and Hanes plots; the mean value was 11.5 mmol/1 for the unstimulated enzyme and 2.8 mmol/1 for the potassium ion-stimulated enzyme. Similar # m -values were found for the unstimulated acid α-glucosidase from human urine (2) and human kidney (12). Potassium ions caused an increased affinity of acid α-glucosidase for maitose, s well s an increased rate of maitose hydrolysis ( fig.…”

“…Glucose concentrations up to 5 mmol/1 in the samples did not disturb the determination of acid α-glucosidase. The activity of acid α-glucosidase is inhibited by tris and by erythritol (12).…”

Acid maltase levels in muscle in heterozygous acid maltase deficiency and in non-weak and neuromuscular disease controls

“…Because purified AM preparations from rat liver (Jeffrey, Brown, and Brown, 1967;Auricchio, Bruni, and Sica, 1968) and from human kidney (Auricchio et al, 1968) were found recently to have Km values of 5 x 10-3M and 13-6 x 10-3M, respectively, with maltose as substrate, it seemed possible that the enzyme in muscle homogenates was suboptimally active at the 5 mM substrate concentration originally recommended by Hers (1963). Maltose hydrolysis was therefore determined with increasing concentrations of maltose in three cases of AM deficiency in adults, in six heterozygotes and in two non-weak and two neuromuscular disease controls (Fig.…”

Characterization of the molecular defect in infantile and adult acid alpha-glucosidase deficiency fibroblasts.

“…She demonstrated progressive muscle weakness and muscle wasting since the age of 30 yr. She died at the age of 47 yr because of respiratory failure. Skin fibroblasts were cultured and harvested at early confluence as described (17 (19). A 4.5 x 45-cm column was used to increase the flow rate.…”