Reconstitution of cell-free NADPH-oxidase from human monocytes and comparison with neutrophils.
A rapid centrifugal elutriation procedure was developed to prepare high amounts of pure human blood monocytes. After disruption by nitrogen cavitation, a cytosol and a membrane fraction were obtained by sucrose-Percoll density centrifugation. The plasma membrane fraction contained cytochrome b558 and was free of microsomal or mitochondrial cytochromes as determined by low-temperature absorbance spectroscopy. Cell-free NADPH-oxidase activity from monocytes and neutrophils was reconstituted with cytosol and membranes in the presence of sodium dodecyl sulphate. By comparison with neutrophils, the cell-free NADPH-oxidase from monocytes showed a two- to threefold lower specific activity. The NADPH-oxidase was reconstituted with neutrophil membranes and monocyte cytosol, and vice versa. The Km for NADPH was always lower when monocyte cytosol was used. These experiments indicate that the membrane-bound components of the NADPH-oxidase from neutrophils and monocyte are similar and that levels of NADPH-oxidase components in the cytosols differ. Interferon gamma-treatment of the monocytes had no effect on the specific activity of the cell-free NADPH-oxidase.
Thelen
M
M
Theodor-Kocher Institute, University of Berne, Switzerland.
Baggiolini
M
M
D002462:Cell Membrane / Q000201:enzymology / Q000648:ultrastructure; D003600:Cytosol / Q000201:enzymology / Q000648:ultrastructure; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D009000:Monocytes / Q000201:enzymology* / Q000648:ultrastructure; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D019255:NADPH Oxidases; D009504:Neutrophils / Q000201:enzymology* / Q000648:ultrastructure
D007371:Interferon-gamma; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases