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In order to study the intracellular localization of the proteolytic processing steps in the maturation of alpha-glucosidase and cathepsin D in cultured human skin fibroblasts we have used incubation with glycyl-L-phenylalanine-beta-naphthylamide (Gly-Phe-NH-Nap) as described by Jadot et al. [Jadot, M., Colmant, C., Wattiaux-de Coninck, S. & Wattiaux, R. (1984) Biochem. J. 219,965-970] for the specific lysis of lysosomes. When a homogenate of fibroblasts was incubated for 20 min with 0.5 mM Gly-Phe-NH-Nap, a substrate for the lysosomal enzyme cathepsin C, the latency of the lysosomal enzymes alpha-glucosidase and beta-hexosaminidase decreased from 75% to 10% and their sedimentability from 75% to 20-30%. In contrast, treatment with Gly-Phe-NH-Nap had no significant effect on the latency of galactosyltransferase, a marker for the Golgi apparatus, and on the sedimentability of glutamate dehydrogenase and catalase, markers for mitochondria and peroxisomes, respectively. The maturation of alpha-glucosidase and cathepsin D in fibroblasts was studied by pulse-labelling with [35S]methionine, immunoprecipitation, polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate and fluorography. When homogenates of labelled fibroblasts were incubated with Gly-Phe-NH-Nap prior to immunoprecipitation, 70-80% of all proteolytically processed forms of metabolically labelled alpha-glucosidase and cathepsin D was recovered in the supernatant. The earliest proteolytic processing steps in the maturation of alpha-glucosidase and cathepsin D appeared to be coupled to their transport to the lysosomes. Although both enzymes are transported via the mannose-6-phosphate-specific transport system, the velocity with which they arrived in the lysosomes was consistently different. Whereas newly synthesized cathepsin D was found in the lysosomes 1 h after synthesis, alpha-glucosidase was detected only after 2-4 h. When a pulse-chase experiment was carried out in the presence of 10 mM NH4Cl there was a complete inhibition of the transport of cathepsin D and a partial inhibition of that of alpha-glucosidase to the lysosomes. Leupeptin, an inhibitor of lysosomal thiol proteinases, had no effect on the transport of labelled alpha-glucosidase to the lysosomes. However, the early processing steps in which the 110-kDa precursor is converted to the 95-kDa intermediate form of the enzyme were delayed, a transient 105-kDa form was observed and the conversion of the 95-kDa intermediate form to the 76-kDa mature form of the enzyme was completely inhibited.(ABSTRACT TRUNCATED AT 400 WORDS)
R P Oude Elferink, and
J Van Doorn-Van Wakeren, and
A Strijland, and
A J Reuser, and
J M Tager
July 1983,
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R P Oude Elferink, and
J Van Doorn-Van Wakeren, and
A Strijland, and
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J M Tager
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R P Oude Elferink, and
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R P Oude Elferink, and
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R P Oude Elferink, and
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R P Oude Elferink, and
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R P Oude Elferink, and
J Van Doorn-Van Wakeren, and
A Strijland, and
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J M Tager
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R P Oude Elferink, and
J Van Doorn-Van Wakeren, and
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R P Oude Elferink, and
J Van Doorn-Van Wakeren, and
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J M Tager
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R P Oude Elferink, and
J Van Doorn-Van Wakeren, and
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