Patterns of amino acid efflux from isolated normal and cystinotic human leucocyte lysosomes.

Abstract

This study describes the first direct measurements of amino acid efflux from human lysosomes.Isolated leucocyte lysosomes can be loaded with radioactive amino acids by exposure to low concentrations of the corresponding labeled amino acid methyl esters.EffIux of amino acid from the loaded lysosomes can then be determined.Conditions during loading are adjusted for each ester to permit its adequate intralysosomal hydrolysis and subsequent accumulation of the free amino acid.Relative rates of efflux were leucine E phenylalanine 2 methionine > tryptophan > > cystine.Efflux of leucine, tryptophan, or cystine was independent of exogenous cation, ATP, or amino acid concentrations under the conditions tested.Leucine efflux was similar in normal and cystinotic lysosomes, providing strong evidence that isolated cystinotic lysosomes do not manifest a generalized defect in amino acid efflux.In both normal and cystinotic lysosomes, cystine efflux was much slower than efflux of leucine or other amino acids from human or rat liver lysosomes.Significant differences in mean cystine efflux between isolated normal and cystinotic lysosomes were not apparent in the present test system, although the possibility of differences in rates could not be excluded.For both fundamental and practical reasons, it would be highly desirable to have a direct method for measuring amino acid efflux from human lysosomes.For example, in the disease known as cystinosis (cystine storage disease) (1,2) a defective efflux mechanism could play a major role in the process leading to intralysosomal cystine accumulation.The molecular defect responsible for cystine storage is not known.Efforts to identify a faulty enzymatic activity which could account for decreased cystine degradation, reduction, or derivatization in cystinotic cells have so far been unsuccessful.Abnormal cystine accumulation could also he due to defective efflux of cystine itself from lysosomes into the cell cytoplasm (1, 2).This possibility has previously been impossible to assess directly.Until recently, the techniques used to study lysosomal permeability have been indirect and were based on the degree of preservation of lysosomal enzyme latency by high concentrations (-0.25 M) of test compounds ( 3 ) .In 1973 Goldman and Kaplan (4) observed loss of lysosomal latency following exposure of isolated rat liver lysosomes to certain L-amino acid methyl esters (0.5-2 mM).The hypothesis emerged from these experiments that the methyl esters, unlike the come-