Use of immobilized antibodies in investigating acid α-glucosidase in urine in relation to Pompe's disease

Abstract

1. (1) A simple method is described for the isolation of the lysosomal enzyme, acid α-glucosidase (α- d-glucoside glucohydrolase, EC 3.2.1.20) from normal human liver. Antibodies raised against the purified enzyme were immobilized by covalent coupling to Sepharose 4B. 2. (2) Acid α-glucosidase can be quantitatively removed from normal urine by incubating with an excess of immobilized antibody. With p- nitrophenyl-α-glucosidase as substrate, acid α-glucosidase accounts for 91 ± 3% of the total α-glucosidase activity at pH 4.0 in normal urine. 3. (3) In urine from a patient with the infantile form of Pompe's disease (‘acid maltase deficiency’), no α-glucosidase activity could be removed by the immobilized antibody, in agreement with the fact that acid α-glucosidase is absent in these patients. 4. (4) In urine from patients with the late-onset form of Pompe's disease, 46 ± 11% of the α-glucosidase activity at pH 4.0 can be removed by incubation with immobilized antibodies, indicating that residual acid α-glucosidase activity is present in urine of these patients. The residual acid α-glucosidase activity amounts to about 5% of that in the urine of control persons. 5. (5) If acid α-glucosidase is adsorbed to immobilized antibodies, the activity can still be measured with p- nitrophenyl-α- glucoside as substrate. The K m for p- nitrophenyl-α- glucosidase is not significantly changed by adsorbing purified acid α-glucosidase to immobilized antibodies. 6. (6) The properties of acid α-glucosidase from urine of patients with late-onset Pompe's disease were compared with those of acid α-glucosidase from normal urine, both adsorbed to immobilized antiserum. The pH-activity profile of the enzyme from urine of patients with late-onset Pompe's disease can not be ditinguished from that of the normal urinary enzyme. The K m for p- nitrophenyl-α-glucosidase of the two enzymes is identical, both at pH 4 and 3. The titration curves of the two enzymes with immobilized antibodies are identical.