Hydrolases in intracellular compartments of rat liver cells. Evidence for selective activation and/or delivery.
We used perfused rat livers to investigate the role of endosomes versus lysosomes in the hydrolysis of endocytosed material. When perfusions were performed at 37 degrees C with 125I-asialoorosomucoid, 125I-galactosylated albumin, or 125I-mannosylated albumin, there was a 15-min lag before trichloroacetic acid-soluble degradation products were detected. Furthermore, no hydrolysis was detected at 16 degrees C, indicating that there was no significant prelysosomal degradation of these proteins. Since detection by this method depends on extensive hydrolysis, we subsequently used three small synthetic molecules from which fluorescent products are generated by a single cleavage. These were 4-methylumbelliferyl sulfate, 4-methylumbelliferyl phosphate, and 4-methylumbelliferyl-beta-D-glucosaminide, which are substrates for aryl sulfatase, acid phosphatase, and beta-hexosaminidase, respectively. Using the first two compounds, hydrolysis was detected after 3 min at 37 degrees C and still occurred, albeit to a reduced extent, at 16 and 4 degrees C. This indicates that aryl sulfatase and acid phosphatase are active prelysosomally. We found a different result with 4-methylumbelliferyl-beta-D-glucosaminide. At 37 degrees C, there was a greater than 15-min lag before hydrolysis products were measured; furthermore, hydrolysis ceased at 16 degrees C, indicating that beta-hexosaminidase is active lysosomally. Taken together, these findings show that there is selective activation and/or delivery of hydrolases along the endocytic pathway.
Casciola-Rosen
L A
LA
Department of Cell Biology and Anatomy, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Hubbard
A L
AL
D000117:Acetylglucosamine / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D001212:Asialoglycoproteins / Q000378:metabolism; D002451:Cell Compartmentation; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism*; D005944:Glucosamine / Q000031:analogs & derivatives / Q000378:metabolism; D006023:Glycoproteins / Q000378:metabolism; D006867:Hydrolases / Q000378:metabolism*; D006923:Hymecromone / Q000031:analogs & derivatives / Q000378:metabolism; D008099:Liver / Q000201:enzymology*; D008247:Lysosomes / Q000201:enzymology; D008297:Male; D010873:Pinocytosis; D051381:Rats; D011919:Rats, Inbred Strains; D012710:Serum Albumin, Bovine / Q000378:metabolism
D001212:Asialoglycoproteins; D006023:Glycoproteins; C019492:4-methylumbelliferyl sulfate; D012710:Serum Albumin, Bovine; C005359:4-methylumbelliferyl phosphate; C025158:4-methylumbelliferyl 2-acetamido-2-deoxy-beta-D-glucopyranoside; D006923:Hymecromone; D006867:Hydrolases; D005944:Glucosamine; D000117:Acetylglucosamine