Gaucher's Disease. I. Solubilization and Electrophoresis of β-Glucosidase from Leukocytes and Cultured Fibroblasts

Abstract

Summary: In order to determine unambiguously the presence of multiple forms of β-glucosidase in normal leukocytes and fibroblasts and the form(s) present in Gaucher's disease, we had to devise techniques to separate β-glucosidase from the lysosomal membrane in a catalytically active form and to visualize the isozymes electrophoreticaily. Separation of relatively stable enzyme from the membrane was not achieved until leukocyte and fibroblast suspensions were freeze-thawed consecutively 10 times. After Cellogel electrophoresis of normal leukocyte extracts, two distinct and discrete bands of β-glucosidase activity, which migrated toward the anode, were observed using the fluorogenic substrate 4-methylumbeIliferyl- β-D-glucopyranoside. In patients with juvenile onset Gaucher's disease, all of the leukocyte β-glucosidase activity remained at the origin. The electrophoretic pattern of β-glucosidase from normal cultured skin fibroblasts showed only one fluorescent band of β-glucosidase activity which also migrated toward the anode, but with lesser mobility than the leukocyte bands. In fibroblast extracts of patients with Gaucher's disease,β-glucosidase activity could not be visualized after electrophoresis, even though prior to electrophoresis these extracts had 15-35% of normal activity.

In contrast to the enzyme from leukocytes and fibroblasts, β- glucosidase activity in the spleen and liver was easily release by homogenization. Only one band of β-glucosidase activity, more rapidaly anodal than the leukocyte isozmes, was ibserved after ekectriohoresis.Enyme acti ity in splenic crude homogenates from five patients with Gaucheresis, disease varied from 5.2-60.8% of normal. After electrophoresis, diminished activety of β-glucosidase with the same mobility as the normal enzyme was detected in the two spleen samples with highest activity.

Speculation: The electrophoretic behavior of leukocyte β-glucosidase from patients with juvenile onset Gaucher'disease suggests that the condition results from the synthesis of an altered enzyme, which is bound enzyme.The abnormal membrane binding could interfere with the accessibility of the enzyme to the natural substrate and account for the gradual accumulation of glycospingolipid in the patients.

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