Molecular characteristics of cytochrome b558 isolated from human granulocytes, monocytes and HL60 and U937 cells differentiated into monocyte/macrophages

Abstract

Enriched cytochrome b 558 preparations were obtained from human mature monocytes (MN) and retinoic acid plus interferon γ induced human myeloid leukemia cell lines HL-60 and U937, using an adaptation of the procedure described by A.W. Segal (Nature (1987) 326, 88–91) for purification of cytochrome b 558 from human polymorphonuclear leukocytes (PMN). Spectral characteristics of cytochrome b 558 were determined and found to be independent of cell type and specific heme b content of the preparation. To increase the sensitivity of the spectral assay, analysis in the γ band were used and Δε 427−413 was determined to be equal to 158 mM −1 cm −1. An αβ type heterodimeric cytochrome b 558 was found for PMN and MN by the concordant elution of heme b spectral activity from heparin agarose and the detection of two polypeptide chains by SDS-PAGE. The expression of the lighter polypeptide α chain in the various human monocyte-like cell lines was assessed and its identity, as a component of cytochrome b, was confirmed by immunodetection using a rabbit polyclonal antibody reacting with the α subunit of PMN cytochrome b 558. Immunoblotting studies detected the α subunit in monocyto-macrophagic differentiated HL-60 and U 937 cells and mature MN at 22 kDa, but not in uninduced cells which did not express the respiratory burst. Whatever the specific content or the cell origin of the cytochrome b 558-enriched preparations, the heme b binding site was shown to be associated with the α subunit defined by a constant molecular mass of 22 kDa, as evidenced by the finding of a constant ratio between the silver stained band intensity and the corresponding heme b amount. The heavy polypeptide β chain from MN cytochrome b was found to have a significantly higher molecular weight than the β subunit from PMN at 94 ± 5 kDa instead of 90 ± 4 kDa. In contrast, in cytochrome b preparations from induced monocyto-macrophagic cells, isolated with a low heme specific content, the variability in the detection of the staining intensity of the β band either in SDS-PAGE or immunodetection reactivities precludes accurate definition of its molecular mass and estimation of the stoichiometry between the α and β subunits in the differentiated cells. However, wheat-germ agglutinin binding studies indicated the presence of N-glycosylated protein in the range of 85–110 kDa.