Incorporation of Glycosylated β‐Galactosidase into Bovine Brain Synaptosomes

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Abstract
Using a synthesized glycoprotein, .beta.-galactosidase modified with p-aminophenyl .beta.-D-galactopyranoside (.beta.-D-Gal .beta.-gal), the incorporation of the glycoprotein into bovine brain synaptosomes was studied. The uptake was mediated by a specific receptor to .beta.-D-galactoside, and was inhibited by GM1 ganglioside. The uptake was found to require energy and to be sensitive to metabolic inhibitors. Kinetic studies on .beta.-D-Gal .beta.-gal uptake indicated the presence of a saturable, carrier-mediated transport system in synaptosomes. By subcellular fractionation the .beta.-D-Gal .beta.-gal taken up was found in the fractions corresponding to the nucleus and membrane fragments, the soluble cytosomal fractions, and the mitochondria and lysosomes. The uptake was markedly increased by addition of Ca2+ to the incubation medium. The maximal uptake was obtained at pH 8.0 in the presence of 10 mM Ca2+ at 37.degree. C. By addition of a Ca2+ ionophore A23187, .beta.-D-Gal .beta.-gal uptake was increased in a dose-dependent way parallel to the increase in the intrasynaptosomal concentration of Ca2+. Preincubation of synaptosomes with calmodulin antagonists such as trifluoperazine and N-(6-aminohexyl)-5-chloro-1-napthalenesulfonamide (W-7) was found to inhibit the uptake markedly, and diazepam, an inhibitor of Ca2+/calmodulin-dependent protein kinase, also inhibited the uptake. At a concentration between 1 and 10 .mu.M, 50% inhibition of the uptake was observed with either inhibitor. On the other hand, the addition of dibutyryl cyclic AMP did not affect the uptake of the glycoprotein into syanptosomes. These results suggest that the incorporation of this macromolecule is dependent on a Ca2+/calmodulin-dependent protein kinase.