Subcellular redistribution of newly synthesized macrophage lysosomal enzymes. Correlation between delivery to the lysosomes and maturation.
Cultured mouse peritoneal macrophages were fractionated by two methods at various times after pulse labeling with [35S]methionine. The lysosomal enzymes beta-glucuronidase and beta-galactosidase were isolated from each fraction by immunoprecipitation and electrophoresis on sodium dodecyl sulfate-acrylamide gels. Two distinct peaks of label were obtained on Percoll density gradients. An early appearing peak of low density, containing the precursor forms of both enzymes, co-sedimented with markers for the endoplasmic reticulum, the Golgi apparatus, and the plasma membrane. With time, immunoprecipitable label cosedimented with the bulk of the lysosomal enzyme activity at high density and corresponded to the mature forms of the lysosomal enzymes. By differential centrifugation, newly synthesized enzymes were found predominantly in small particle fractions, unlike the bulk of the lysosomal enzymic activity which was found in larger particle fractions. With increasing time, newly synthesized enzymes were transferred to assume a distribution similar to that of lysosomal enzymic activity. The results suggest that transport of newly synthesized enzymes to lysosomes and conversion to mature forms are closely linked events. Conversion of lysosomal precursors to mature forms occurs either in a prelysosomal vesicle or shortly after reaching the lysosome. The two enzymes follow similar subcellular pathways at similar rates. Also, the macrophage system appears suitable for direct analysis of newly synthesized lysosomal enzymes during subcellular transport.
D000818:Animals; D002499:Centrifugation, Density Gradient; D005260:Female; D005966:Glucuronidase / Q000378:metabolism; D008247:Lysosomes / Q000201:enzymology*; D008264:Macrophages / Q000201:enzymology*; D051379:Mice; D008810:Mice, Inbred C57BL; D013347:Subcellular Fractions / Q000201:enzymology; D014018:Tissue Distribution; D001616:beta-Galactosidase / Q000378:metabolism
D001616:beta-Galactosidase; D005966:Glucuronidase