Accumulation of amino acids within intracellular lysosomes of rat polymorphonuclear leukocytes incubated with amino acid methyl esters. Evidence for the internal acidification of azurophilic granules.

Rat peritoneal polymorphonuclear leukocytes accumulate radioactivity when incubated with 17 microM L[4,5-3H]leucine methyl ester; only low levels of accumulation are observed when the cells are incubated with equivalent amounts of labeled D-leucine methyl ester or the free amino acid, L-leucine. More than 98% of the intracellular radioactivity of cells incubated with leucine methyl ester is in the form of leucine. When cells that had been preincubated with L-leucine methyl ester were disrupted by sonication, 72% of the intracellular radioactivity could be trapped on glass fiber filters, indicating that the leucine had accumulated within a particulate intracellular compartment. Granule preparations obtained from the polymorphonuclear leukocytes accumulated leucine in the presence of leucine methyl ester in a manner similar to the intact cells. Separation by density gradient centrifugation of the granules obtained from cells preincubated with labeled leucine methyl ester revealed a nearly exact correspondence between the density distribution of accumulated radioactivity and that for beta-glucuronidase, an enzyme localized with the azurophilic subpopulation of polymorphonuclear leukocyte granules; there was no such correspondence with the activity of alkaline phosphatase, a marker for the specific granules. The results indicate that the azurophilic granules represent the intracellular site of leucine accumulation by the intact cells. This conclusion is supported by the effects of millimolar concentrations of amino acid methyl ester, which cause a dramatic loss of latency for the activities of the azurophilic granule enzymes beta-glucuronidase and myeloperoxidase but have no effect upon the latency of the specific granule enzyme alkaline phosphatase. Leucine accumulation by the intact cells is 80% inhibited by 0.1 mM chloroquine and 50% inhibited by 20 mM NH4Cl and methylamine, lysosomotropic agents that elevate the intralysosomal pH. These observations suggest that the azurophilic granules of these cells, generally considered to be primary lysosomes, are internally acidified.