BIOSYNTHESIS, POST-TRANSLATIONAL PROCESSING AND PACKAGING OF MYELOPEROXIDASE INTO AZUROPHIL GRANULES IN HL-60 CELLS (LYSOSOMES, LEUKEMIA)

Myeloperoxidase is a heme-containing protein comprised of 2 large (ca. 60K) and 2 small (ca. 14K) subunits. It is produced in promyelocytes and packaged into cytoplasmic azurophil granules. In addition, these granules contain many enzymes commonly found in lysosomes and are generally considered specialized lysosomes. The purpose of this dissertation was to study biosynthesis, post-translational processing and packaging of myeloperoxidase in azurophil granules in promyelocytic HL-60 cells and to compare these processes to those which have been detailed by lysosomal enzyme synthesis and transport.;Myeloperoxidase was produced as an 81K polypeptide which was cotranslationally modified to a 91K species by the addition of high mannose carbohydrate. This species was either constitutively secreted by the cell or modified first to an 81K intracellular intermediate and then to a 74K intermediate by neutral serine proteinases contained within clathrin-coated vesicles. The 74K intermediate was the immediate precursor to mature, large subunits of myeloperoxidase in crude granule extracts. Production of mature myeloperoxidase was linked to its deposition into dense granules.;All proteolytic events were found to be acid independent; however, the addition of a Na('+), protonophore, monensin, to cells in culture resulted in a >40% inhibition of mature myeloperoxidase production over a 20 hour period. In addition, increased levels of these species were localized to light density organelles which co-migrated with Golgi and endoplasmic reticulum identified both biochemically and ultrastructurally, suggesting that monensin treatment blocked transport of myeloperoxidase into maturing granules. Increased levels of immature levels of immature species in monensin-treated cells required acid pH for extraction from granule fractions, suggesting their association with an acid-dependent receptor similiar to the mannose-6-phosphate receptor responsible for targeting in the lysosomal system.;The immature forms present in monensin-treated cells were not associated with identifiable Golgi structures although ultrastructural effects were essentially limited to that organelle. Instead, these forms were located in clathrin-coated vesicles. Histochemical staining patterns of monensin-treated cells suggest that certain Golgi-derived properties were decreased in azurophil granules and that small vesicles containing immature protein accumulated and fused in the perinuclear region. (Abstract shortened with permission of author.).