Abstract
Lysosomes contain enzymatic activities capable of degrading nucleic acids to their constituent nucleosides, but the manner by which these degradation products are released from the lysosome is unknown. To investigate this process, human fibroblast lysosomes, purified on Percoll density gradients, were incubated with [3H]adenosine at pH 7.0, and the amount of adenosine taken up by the lysosomes was measured. Adenosine uptake by fibroblast lysosomes attained a steady state by 12 min at 37 degrees C and was unaffected by the presence of 2 mM MgATP or changes in pH from 5.0 to 8.0. An Arrhenius plot was linear with an activation energy of 12.9 kcal/mol and a Q10 of 2.0. Lysosomal adenosine uptake is saturable, displaying a Km of 9 mM at pH 7.0 and 37 degrees C. Various nucleosides and the nucleobase, 6-dimethylaminopurine, strongly inhibit lysosomal adenosine uptake, whereas neither D-ribose or nucleotide monophosphates have any significant effect upon lysosomal adenosine uptake. On a molar basis, purines are recognized more strongly than pyrimidines. Changing the nature of the nucleoside sugar from ribose to arabinose or deoxyribose has little effect on reactivity with this transport system. The known plasma membrane nucleoside transport inhibitors, dipyridamole and nitrobenzylthioinosine, inhibit lysosomal nucleoside transport at relatively low concentrations (25 microM) relative to the Km of 9 mM for lysosomal adenosine uptake. The half-times of [3H]inosine and [3H]uridine efflux from fibroblast lysosomes ranged from 6 to 8 min at 37 degrees C. Trans effects were not observed to be associated with either inosine or uridine exodus. In contrast to adenosine uptake, adenine primarily enters fibroblast lysosomes by a route not saturable by high concentrations of various nucleosides. In conclusion, the saturability of lysosomal adenosine uptake and its specific, competitive inhibition by other nucleosides indicate the existence of a carrier-mediated transport system for nucleosides within fibroblast lysosomal membranes.
Highlights
Lysosomal adenosine uptake studies have allowed us to describe a carrier-mediated transport system serving for the passage of nucleosides across the lysosomal membrane of
Lysosomal Adenosine Exodus Studies-Percoll-purified fibroblast lysosomes were incubated for 20 min at 37 "C in a volume of 0.34 ml trifugation through Percoldl ensity gradients
A, double-reciprocalplot of the initial rate of t3H]adenosine uptake as a function of the adenosine concentration for the different concentrations of inosine which were lysosomal adenosine uptake by inosine was calculated to be 14 mM as determined by Cleland’sCOMP program [27]. Such an analysis was performed for a variety of analogs, all of which were found to be competitive inhibitors of lysosomal adenosine uptake
Summary
Incubated for 2.5 min at 37 "C;a 34-p1aliquot was removed from Lysosomes werepurifiedfromhuman fibroblasts by ceneach incubation mixture, added to 12 ml of ice-cold phosphatebuffered saline, and filtered through a GF/A glass fiber filter as described above. Lysosomal Adenosine Exodus Studies-Percoll-purified fibroblast lysosomes were incubated for 20 min at 37 "C in a volume of 0.34 ml trifugation through Percoldl ensity gradients. J incubated a t 37 "C; 50-pl aliquots, removed at the indicated time points, were added to 12 ml of ice-cold phosphate-buffered saline and filtered through GF/A filters as described above. At the indicated time points, is defined as the amount of enzyme activity necessary to produce 1 aliquota were removed from the incubation mixture, lysosomes were nmol of p-nitrophenollmin at 37 'C in the above assay.
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