Isolation of NADPH oxidase from murine peritoneal macrophages: a comparison of methods

Abstract

302BIOCHEMICAL SOCIETY TRANSACTIONS level for both bound and ingested counts. Significantly greater number of galactosamine microspheres were ingested than any other labelled type.Electron microscopic autoradiography revealed that was incorporated at both the apical and basal cell surfaces but not intracellularly. Forty-eight proteins were labelled with "<I (Fig. 1). Two protein spots ( 2 5 and 26 on Fig. 1) were enhanced in intensity after challenge with labelled microspheres when compared with unlabelled spheres and the unchallenged control.These results indicate that the W E cells are able to differentiate between labelled and unlabelled phagocytic particles and also to recognize specific ligands on target particles. The increase in synthesis of the plasma membrane protein doublet (which may represent a recognition component) in response to specific challenges only, supports the view that a receptor-mediated mechanism is involved in the phagocytosis of ROS and some other biotic particles by the RPE but that a non-specific mechanism is involved in phagocytosis of inert particles.We are grateful to the British Retinitis Pigmentosa Society for financial support. We are also grateful to BioRad laboratories for their contribution towards the cost of attending this Meeting.Phagocytic cells are major effectors of the human immune system and are known to produce oxygen radicals in a socalled respiratory burst as part of their toxicity towards micro-organisms. A large number of agents are capable of stimulating respiratory burst activity, e.g. phorbol myristate acetate, lipopolysaccharide and zymosan. The effect of these stimuli is greatly enhanced when they are applied to cells previously activated in vivo.The production of the superoxide anion (0,) is the first stage of the respiratory burst and leads to formation of hydrogen peroxide (H,O,) and the hydroxyl radical ('OH).These oxygen radicals are known to produce widespread effects in body tissues. In the case of macrophages, powerful tissue-based phagocytes, these radicals are directed principally against invasive pathogens and tumour cells. A sideeffect of such activity may, however, be the initiation of damage to normal healthy tissues.After a suitable stimulus, macrophages produce 0 ; by the one-electron reduction of molecular oxygen at the expense of NADPH. The reaction is catalysed by the membrane-bound enzyme NADPH oxidase [ 11. NADPH oxidase is believed to comprise a mini electron transport chain, distinct from the mitochondria1 redox chain, consisting of a flavoprotein, a b-cytochrome and possibly a quinone [ 31.Isolation of the active form of NADPH oxidase has been achieved by a variety of methods. The usual source of the enzyme has been peripheral blood granulocytes. In this study we attempted to isolate NADPH oxidase from macrophages activated in vivo. A comparison was made of three available methods: (a) Percoll-density gradient centrifugation of isolated cell membranes and formation of solubilized membrane suspensions [4], (b) hypertonic sucrose-densit...