Transport of D-glucose was studied in Percoll-gradient-purified rat liver lysosomes. D-Glucose uptake had a Km of 22 mM and a t1/2 of approx. 30 s. D-Fucose, 2-deoxyglucose and methyl alpha-glucoside were the most effective competitors for uptake of D-glucose, although D-galactose, D-mannose, D-xylose and L-fucose also appeared to compete for uptake. L-Glucose was a poor competitor for uptake. No competition was observed with N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, D-glucuronic acid, N-acetylneuraminic acid, D-glucosamine or the amino acids L-glycine, L-lysine and L-proline. Uptake was unaffected by N-ethylmaleimide, dithiothreitol, KCl, NaCl, ATP/Mg or alteration of buffer pH. D-Glucose efflux from lysosomes was temperature-dependent, with a Q10 of 2.3, and was inhibited by cytochalasin B. Counter-transport could not be demonstrated. In contrast, L-fucose uptake had a Km of 65 mM and was largely unaffected by 5 M excess of neutral D-sugars. Both uptake and efflux of L-fucose were inhibited by cytochalasin B. It appears that lysosomes possess a facilitated transport system for D-glucose and perhaps other neutral D-sugars that is discrete from transport systems for acetylated and acidic sugars.

Countertransport of GlcNAc was examined in membrane vesicles prepared from rat liver lysosomes which had been lysed by exposure to 5 mM-methionine methyl ester. These vesicles have a random orientation, have intact ATP-dependent acidification and are largely free of soluble hydrolases. Vesicular volume (24.69 +/- 4.51 microliters/mg of protein) was greater than that of lysosomes (3.02 +/- 0.56 microliters/mg of protein), corresponding to a doubling of diameter. Characteristics of GlcNAc transport in vesicles (Km = 1.3 mM) were similar to those observed in intact lysosomes (Km = 4.4 mM). Sulphation or phosphorylation of the substrate resulted in loss of recognition by the carrier. Hydroxyl group orientation at multiple positions did not appear to be critical, whereas orientation of the acetyl group appeared to have a fundamental role in recognition by the carrier. Based on these criteria, phenyl isothiocyanate-GlcNAc (PITC-GlcNAc) was identified as a possible substrate for transport. Under mild conditions, PITC-GlcNAc reversibly inhibited GlcNAc countertransport in lysosomes and vesicles. This and other modified substrates may be of value in identification of the GlcNAc/GalNAc lysosomal transporter. Lysosomal membrane vesicle preparation is a technique that should be useful for the study of other lysosomal transport systems.

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