Subcellular distribution of acid alpha-glucosidase in fibroblasts and of antigenically cross-reactive material in Pompe's disease fibroblasts.
From fibroblasts of two cases of Pompe's disease (acid alpha-glucosidase deficiency), one of the childhood type (RH-SF-1) and one of the adult type (RH-SF-2), and normal fibroblasts, antigenically cross-reactive material and acid alpha-glucosidase were immunoprecipitated and analysed by immunoelectrotransfer blotting. The acid alpha-glucosidase and antigenically cross-reactive material (which reacts with antibody raised against normal acid alpha-glucosidase) revealed a precursor form of molecular weight 97,000 and two major components of 79,000 and 76,000. When monensin was added to the fibroblast culture, the two major components of normal acid alpha-glucosidase were decreased, whereas the large molecular weight precursor was increased. On the other hand, the 97,000 molecular weight component of cross-reactive material in the Pompe's fibroblasts (RH-SF-1 and RH-SF-2) was only slightly increased on monensin treatment. The fibroblasts were pulse-chase labelled with [2-H3] mannose and 32Pi. The cross-reactive material and acid alpha-glucosidase were precipitated with anti acid alpha-glucosidase antibody, and after sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), fluorography was performed. The radiolabel of 3H in the cross-reactive material of RH-SF-1 and -2 was weak, and 32P in the cross-reactive material of both fibroblasts was very weak when compared with those of the acid alpha-glucosidase. The radiolabel of 32P in the cross-reactive material of RH-SF-1 was extremely weak. Immunofluorescence histochemistry revealed a granular localization of acid alpha-glucosidase in the normal fibroblast cytoplasm, and a diffuse distribution of cross-reactive material in the cytoplasm of RH-SF-1 and -2. Immuno-electron microscopic examinations showed a normal acid alpha-glucosidase localization on the inner side of the lysosomal membrane and also diffusely in the lysosome; when treated with monensin, it was present on the trans part of the Golgi apparatus. Antigenically cross-reactive material, however, was found in the cytoplasm and endoplasmic reticulum. Some lysosomal localization was observed sporadically. Even after monensin treatment, it was not demonstrated on the Golgi apparatus.
D000941:Antigens / Q000276:immunology; D002648:Child; D003593:Cytoplasm / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000201:enzymology; D005347:Fibroblasts / Q000187:drug effects / Q000201:enzymology / Q000648:ultrastructure*; D005455:Fluorescent Antibody Technique; D006008:Glycogen Storage Disease / Q000201:enzymology*; D006009:Glycogen Storage Disease Type II / Q000201:enzymology*; D006056:Golgi Apparatus / Q000201:enzymology; D006651:Histocytochemistry; D006801:Humans; D007163:Immunosorbent Techniques; D008247:Lysosomes / Q000201:enzymology; D008297:Male; D008358:Mannose / Q000378:metabolism; D008854:Microscopy, Electron; D008875:Middle Aged; D008970:Molecular Weight; D008985:Monensin / Q000494:pharmacology; D010710:Phosphates / Q000378:metabolism; D011498:Protein Precursors / Q000378:metabolism; D000520:alpha-Glucosidases / Q000276:immunology / Q000378:metabolism*
D000941:Antigens; D010710:Phosphates; D011498:Protein Precursors; D008985:Monensin; D000520:alpha-Glucosidases; D008358:Mannose