Myeloperoxidase precursors incorporate heme.
Myeloperoxidase of neutrophil granulocytes is synthesized as a larger molecular weight precursor, which is processed to yield mature polypeptides with molecular weights of 62,000 and 12,000. We have investigated the incorporation of heme into myeloperoxidase of the human promyelocytic HL-60 cell line labeled with 5-amino[14C]levulinic acid. Myeloperoxidase was isolated by immunoprecipitation followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and radiolabeled myeloperoxidase was visualized by fluorography. A 3-h pulse labeling with 5-amino[14C]levulinic acid resulted in labeling of the Mr 90,000 and Mr 82,000 precursor polypeptides. During subsequent chase of the label, conversion to mature radioactive heavy Mr 62,000 subunit was observed but no radioactivity was associated with the mature small Mr 12,000 subunit. Peptide mapping after proteolytic cleavage with V8 proteinase showed that 5-amino[14C]levulinic acid was associated with a single Mr 23,000 polypeptide while multiple radioactive fragments were visible after proteolytic cleavage of myeloperoxidase biosynthetically labeled with [14C]leucine. That 5-amino[14C]levulinic acid was specifically incorporated into heme of myeloperoxidase was also demonstrated by dissociation under reducing conditions which yielded 14C-labeled heme as indicated by reversed phase high pressure liquid chromatography. The ionophore monensin and the base chloroquine, which block processing of myeloperoxidase, did not affect the incorporation of 5-amino[14C]levulinic acid, further supporting the notion that the incorporation of heme is independent of final processing of the polypeptide. Our data establish that heme is incorporated into myeloperoxidase already at the level of the precursor and that processing yields a heme-containing heavy subunit and a heme-free small subunit.
D000622:Aminolevulinic Acid / Q000378:metabolism*; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism; D006098:Granulocytes / Q000201:enzymology*; D006418:Heme / Q000378:metabolism*; D006801:Humans; D007163:Immunosorbent Techniques; D007930:Leucine / Q000378:metabolism; D007982:Levulinic Acids / Q000378:metabolism*; D008970:Molecular Weight; D010446:Peptide Fragments / Q000378:metabolism; D009195:Peroxidase / Q000378:metabolism*; D011498:Protein Precursors / Q000378:metabolism*; D012697:Serine Endopeptidases
D007982:Levulinic Acids; D010446:Peptide Fragments; D011498:Protein Precursors; D006418:Heme; D000622:Aminolevulinic Acid; D009195:Peroxidase; D010450:Endopeptidases; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D007930:Leucine