| Myeloperoxidase (EC 1.11.1.7, MPO), a hemoprotein synthesized in the endoplasmic reticulum (ER) of human neutrophils as a single-chain precursor, undergoes complex post-translational modifications prior to azurophilic granule packaging. Succinylacetone (SA), a potent inhibitor of heme biosynthesis, demonstrated the effect of heme availability on MPO maturation in human promyelocytic leukemia HL-60 cells. 250 ;Trypsin treatment of radiolabeled HL-60 cell MPO demonstrated protease-resistant fractions of precursor and intermediate MPO in the post-granular supernatant and granules, respectively, suggesting that a conformational change occurred during post-translational processing. Low-temperature incubations indicated that precursor MPO passed through both the ER and the Golgi apparatus.;The potential for successful MPO maturation and storage granule targeting in non-myeloid secretory cells was determined by MPO cDNA transfection into mouse pitiutary tumor AtT20 cells. Expressed MPO proved to be the inactive precursor species, which was gradually secreted into the medium. Immunocytofluorescence revealed particulate MPO aggregation resembling that of ACTH, a pitiutary protein found in regulated secretory granules, and diffuse cytoplasmic MPO fluorescence. Percoll-sucrose fractionation resolved 51% of the MPO in a compartment localized at the same density as lysosomal enzymes, yet distinct from ACTH vesicles. Stimulation with the secretagogue 8Br-cAMP suggested that MPO secretion was primarily constitutive rather than regulated through storage granules. Immunoreactive MPO was demonstrated in an organelle unlike ACTH dense core secretory vesicles.;Investigation of MPO processing in immature high passage HL-60 cells showed decreased MPO activity and a 2.7-fold reduction in mature MPO production. Taken together, these studies supplement knowledge of the complex post-translational processing and targeting systems for MPO, and suggest that they may be myeloid-specific. |
