Andrew R. Cross

ENZYMIC MECHANISMS OF SUPEROXIDE PRODUCTION

The realization that superoxide, and other reactive oxygen species, is produced in significant quantities in biological systems is a relatively recent one and the nature and purpose of the radical-generating systems is in most cases, only beginning to be understood. It is the purpose of this review to describe the known biological sources of superoxide, the enzymic machinery used to generate it, and the possible functions to which it is put

Inhibitors of the leukocyte superoxide generating oxidase: Mechanisms of action and methods for their elucidation

Oxygen radical production by phagocytic cells is currently receiving a great deal of attention as the role of radical damage is becoming apparent in inflammatory diseases, reperfusion injury, cancer, and aging. A large number of inhibitors of the superoxide generating oxidase are known, including standard and experimental anti-inflammatory and anti-rheumatic drugs, natural products, anaesthetics, tranquillizers and antibiotics, in addition to compounds used as experimental tools. The composition of the oxidase and the possible sites of inhibition of these compounds are discussed together with the possible mechanisms of activation of the oxidase and the effects these agents may have on these pathways. Use of these compounds has provided a great deal of information about the components and the nature of the activation processes involved in the stimulation of radical production by leukocytes, as well as pointing to possible targets for the production of novel anti-inflammatory agents. It is clear however that further understanding of the precise nature of the activation pathways and the extent of the involvement of leukocyte-derived oxygen radicals in disease processes will require more specific inhibitors than most of those currently available.

The cytochrome b and flavin content and properties of the O2−-forming NADPH oxidase solubilized from activated neutrophils

NADPH-dependent O2- -generating activity was extracted and partially purified from guinea pig polymorphonuclear leukocytes. The most active preparation generated 202.8 nmol O2- min/min per mg protein. This activity was 30-fold higher than that of extracts from resting cells, indicating that the activated state of the oxidase was retained after solubilization. The solubilization and purification of the enzyme activity were followed by a parallel solubilization and purification of cytochrome b. Spectroscopic studies showed that solubilized cytochrome b has an Em of -245 mV and binds CO to about 30%. Cytochrome b was reduced by NADPH in anaerobiosis at a low rate and was rapidly reoxidized by air. A correlation was found between the inhibition of O2- formation caused by the SH reagent p-chloromercuribenzoate and the alterations induced by this compound on the Em of cytochrome b. These observations strongly support the participation of cytochrome b in the catalytic activity of the solubilized NADPH oxidase. The enzyme preparations contained FAD, which was found to be associated both with NADPH oxidase and with diaphorase activities. The fraction with the highest O2- forming activity contained FAD and cytochrome b in a ratio of about 0.5:1. The participation of FAD in the electron transport from NADPH to O2 is supported also by the inhibitory effect exerted by quinacrine on O2- formation.

A variant form of X-linked chronic granulomatous disease with normal nitroblue tetrazolium slide test and cytochrome b

Abstract. Chronic granulomatous disease was diagnosed in a boy who suffered from severe generalized infections. Family investigations revealed the inheritance of the disease to be X‐linked. However, unlike other cases of X‐linked chronic granulomatous disease, the membrane oxidase of the neutrophils from this patient was not totally defective and sufficient activity was left to result in a normal phorbol myristate acetate‐stimulated nitroblue tetrazolium slide test. Also, unlike the usual findings in X‐linked chronic granulomatous disease, cytochrome b was present in normal amounts in the neutrophils from this patient. The cytochrome was normal, judged from its midpoint potential of —245 mV and its ability to bind CO. It is thus apparent that X‐linked chronic granulomatous disease may result from at least two different defects and that the phorbol myristate acetate stimulated nitroblue tetrazolium slide test fails to detect some cases.

NADPH oxidase-like activity in rainbow trout Oncorhynchus mykiss (Walbaum) macrophages.

Evidence for the existence of an NADPH oxidase-like enzyme in rainbow trout macrophages is given. Reduced-minus-oxidised difference spectroscopy revealed the presence of a cytochrome b with three absorbance peaks, at 430, 533, and 558 nm. The low midpoint potential of the latter peak suggests this cytochrome is the same as the terminal component of NADPH oxidase (i.e., cytochrome b-245). Subcellular fractionation of macrophages revealed two peaks of cytochrome b activity, in accord with the concept of a plasma membrane localisation of cytochrome b activity in addition to a mitochondrial localisation. Finally, that the rainbow trout oxidase is a multicomponent enzyme was suggested by inhibitor studies, where specific inhibitors of the flavin and cytochrome b-245 components of NADPH oxidase induced significant reduction in superoxide anion production.

The use of diphenylene iodonium, an inhibitor of NADPH oxidase, to investigate the antimicrobial action of human monocyte derived macrophages.

Diphenylene iodonium is an inhibitor of the respiratory burst-generating NADPH oxidase of phagocytes. The effect of this compound on human monocyte-derived macrophages and its usefulness in exploring the antimicrobial mechanisms of phagocytes was examined. 1 microM diphenylene iodonium inhibited hydrogen peroxide production by human macrophages and the activity of these cells against Toxoplasma gondii. At this concentration macrophage degranulation was unaffected.

The nature and function of the microbicidal oxidase system of neutrophils.

Stimulated neutrophils demonstrate a burst of nonmitochondrial respiration.1,2 This appears to be important for the killing of certain bacteria which are killed less efficiently under anaerobic conditions,3 or by cells from patients with Chronic Granulomatous Disease (CGD) which fail to demonstrate this respiratory burst.4

CHARACTERISATION OF THE NADPH - DEPENDENT SUPEROXIDE - GENERATING OXIDASE OF PHAGOCYTIC LEUKOCYTES

Phagocytic leukocytes play a major role as part of the immune system in providing protection against infection. As part of their microbicidal activity, neutrophils, the most abundant blood phagocytes, convert molecular oxygen to highly reactive and toxic derivatives such as the superoxide anion, \({{O}_{2}}^{\overline{\centerdot }}\), hydrogen peroxide, H2O2, hypochlorite, OCl−, the hydroxyl radical, OH , and possibly singlet oxygen, 1O2, (1).