PMID: 2112033Jun 1, 1990Paper

Reconstitution of cell-free NADPH-oxidase from human monocytes and comparison with neutrophils.

Blood
M Thelen, M Baggiolini

Abstract

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.

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