Assembly of dimeric myeloperoxidase during posttranslational maturation in human leukemic HL-60 cells.
Myeloperoxidase is a major protein component of the azurophilic granules (specialized lysosomes) of normal human neutrophils and serves as part of a potent bactericidal system in the host defense function of these cells. In normal, mature cells, myeloperoxidase occurs exclusively as a dimer of Mr 150,000 while in immature leukemia cells, there are both monomeric (Mr 80,000) as well as dimeric species. Like other lysosomal enzymes, myeloperoxidase is synthesized as a larger glycosylated precursor (Mr 91,000) that undergoes processing through single-chain intermediates (Mr 81,000 and 74,000) to yield mature heavy (Mr 60,000) and light (Mr 15,000) subunits. To study the assembly of dimeric myeloperoxidase, azurophilic granules were isolated from either unlabeled or pulse-labeled ([35S]methionine/cysteine) HL-60 cells, and myeloperoxidase was extracted and separated into monomeric and dimeric forms by FPLC gel filtration chromatography. Steady-state levels of dimeric and monomeric myeloperoxidase were found to account for 67% and 33%, respectively, of the total peroxidase activity and were correlated with the levels of associated heme as measured by absorption at 430 nm. Labeled myeloperoxidase polypeptides were immunoprecipitated using a monospecific rabbit antibody and were identified and quantitated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis/fluorography and liquid scintillation counting. After a 2-h pulse, labeled myeloperoxidase species of Mr 74,000 and 60,000 were found in fractions coeluting with the monomeric form of myeloperoxidase.(ABSTRACT TRUNCATED AT 250 WORDS)
Taylor
K L
KL
Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322.
Guzman
G S
GS
Burgess
C A
CA
Kinkade
J M
JM
D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006418:Heme / Q000032:analysis; D006801:Humans; D007700:Kinetics; D007938:Leukemia / Q000201:enzymology*; D046911:Macromolecular Substances; D008970:Molecular Weight; D009195:Peroxidase / Q000378:metabolism*; D011233:Precipitin Tests; D011499:Protein Processing, Post-Translational; D014407:Tumor Cells, Cultured
D046911:Macromolecular Substances; D006418:Heme; D009195:Peroxidase