Transport of N-acetyl-D-glucosamine and N-acetyl-D-galactosamine by Rat Liver Lysosomes

A J Jonas,R J Speller,P B Conrad,W P Dubinsky

doi:10.1016/s0021-9258(18)83683-3

A J Jonas, R J Speller + Show 2 more

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https://doi.org/10.1016/s0021-9258(18)83683-3

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Abstract

Transport of N-acetyl-D-glucosamine and N-acetyl-D-galactosamine, products of lysosomal glycoprotein and glycosaminoglycan degradation, was examined in Percoll gradient purified rat liver lysosomes. Uptake of these two sugars was competitive and quite specific remaining largely unaffected by the presence of L-fucose, D-glucosamine, D-glucose, D-glucuronic acid, D-mannose, or N-acetylneuraminic acid. Kinetic studies revealed a Km of 4.4 mM for both N-acetyl-D-glucosamine and N-acetyl-D-galactosamine uptake. Temperature dependence studies revealed a Q10 of 2.3. N-Acetyl-D-glucosamine uptake was not dependent upon NaCl, KCl, MgCl2, or ATP/MgCl2 and was unaffected by 5 mM dithiothreitol or variation of buffer pH between 6.0 and 8.0. Cytochalasin B at a concentration of 50 microM effectively inhibited uptake of N-acetyl-D-glucosamine by 90% and N-acetyl-D-galactosamine by 65%. Prior incubation of lysosomes in 20 mM N-acetyl-D-glucosamine stimulated uptake of both sugars 3-4-fold, although it had no effect on the uptake of D-glucose. Countertransport was unaffected by neutral and cationic amino acids demonstrating independence from these amino acid transport systems. We conclude that lysosomes possess a highly specific transport system for N-acetyl-D-glucosamine and N-acetyl-D-galactosamine.

Highlights

From the Departmentsof Pediatrics and of ‘$Physiologyand Cell Biology, University of Teras Medical School, Houston, Texas 77030
We report our findings of a specific transtem for N-acetyl-D-glucosamine aNnd-acetyl-D-gdac- porter for these two acetylated amino sugars in rat liver tosarnine
Similar results were obtained for the uptake of N-aCetyl-D-galaCtOSamine.Variation of substrate concentrationfrom 1 to 20 mM demonstrated saturation kinetics for uptake with a K, of 4.4 mM for both N-acetyl-D-glucosamine and N-acetyl-D-galactosamine (Fig. 2)

Summary

EXPERIMENTAL PROCEDURES

Several lysosomal transport systems for small molecular weight compounds have been identified in recent years. Amino acids, vitamin BI2,and possibly inorganic compounds generated by the enzymatic degradation of larger structures exit the lysosomes and become available for recycling within the cell [1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18].Studies of amino acid transport have revealed sodium-independent systems for cystine, neutral amino acids, and cationic amino acids which are apparently discrete from plasma membrane systems [1,2,3,4,5,6,7,8] These systems exhibit countertransport and are affected by changes in pH. Countertransport Experiments-Lysosomes still in Percoll were incubated with 20 mM N-acetyl-D-glucosaminefor 1h at 25 "C They were diluted 20-fold with ice-cold buffer (0.25 M sucrose, 20 mM Hepes, pH 7.0) and were collected by centrifugation for 10 min at 13,000 X g. Protein was acid-precipitated by the addition of 10 ql of 50% sulfosalicylicacid to thesonicate

RESULTS

50 PM cytochalasin B 20 mM79L-glycine 20 mM8L6-leucine

DISCUSSION