Akin D T, Kinkade J M, Parmley R T
Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322.
Arch Biochem Biophys. 1987 Sep;257(2):451-63. doi: 10.1016/0003-9861(87)90590-x.
The biosynthesis of myeloperoxidase in human promyelocytic leukemia HL-60 cells was studied by pulse-chase and immunoprecipitation methods and separation of subcellular organelles using Percoll density gradient fractionation. These studies revealed that in control and monensin (1 microM) treated cells, more than 85% of the total immunoprecipitable radiolabeled myeloperoxidase was present predominantly in precursor form (Mr 91,000) and resided in lower density compartments after an initial 3-h labeling period. Using biochemical and ultrastructural techniques, the lower density regions of the gradient were found to contain elements of the endoplasmic reticulum and the Golgi complex. Following a 16-h chase period, about 70% of the radiolabeled myeloperoxidase in untreated cells was found predominantly in denser regions of the gradient and was present mainly in the form of the mature large subunit (Mr 63,000). These dense regions were shown to contain azurophilic granules by means of the distribution of beta-glucuronidase and myeloperoxidase activities and by electron microscopy. Processing of myeloperoxidase and its deposition into dense granules were blocked by monensin treatment. Following a 16-h chase period in the presence of monensin, approximately 80% of the radiolabeled myeloperoxidase continued to reside in lower density compartments and was predominantly in precursor (Mr 91,000) and intermediate (Mr 81,000 and 74,000) forms. Only about 10% of the radiolabeled myeloperoxidase was associated with dense azurophilic granules. Monensin treatment produced large, Golgi-derived vacuoles which were isolated using Percoll density centrifugation and identified by electron microscopy. These vacuoles were found to be essentially devoid of peroxidase activity and pulse-labeled, newly synthesized radiolabeled myeloperoxidase species. The effects of monensin on transport and processing were reversible after a 3-h exposure and 16-h chase period in the absence of monensin. Taken together, these data indicate that maturation of myeloperoxidase is closely linked to its deposition into dense azurophilic granules via a monensin-sensitive process(es). The lower density compartments within which immature myeloperoxidase species accumulate in the presence of monensin appear to be functionally related to or associated with Golgi or endoplasmic reticulum structures distinct from the large monensin-induced vacuoles.
