Activation of the guinea pig granulocyte NAD(P)H-dependent superoxide generating enzyme: localization in a plasma membrane enriched particle and kinetics of activation

Abstract

The subcellular localization, kinetics of activation, and substrate specificity of the guinea pig granulocyte superoxide (O2-) generating system was investigated. Membrane-enriched particles (podosomes) were made from granulocytes by mild sonication and differential centrifugation. These podosomes are enriched threefold for known plasma membrane markers, 5′-nucleotidase, and adenylate cyclase. Podosomes made from resting granulocytes have very little NAD(P)H-dependent O2- production. Podosomes made from cells stimulated with digitonin are equally enriched for membrane markers but have a 15- to 20-fold increase in NAD(P)H-dependent O2- production. The KmAPP for NADPH is one-tenth that for NADH, but the Vmax is the same. The kinetics of digitonin-stimulated whole-cell O2- production parallel the changes in enzyme activity in these podosomes. Temperature affects both the rate and extent of activation of this enzyme. The pH optimum for the enzyme, the pH optimum for activation, and the pH optimum for whole-cell O2- production are all 7.5. Enzyme activity is increased if the cells are treated with glucose and cyanide, inhibited in cells treated with 2- deoxyglucose (2-DOG), and requires the presence of calcium for activation. These effects are similar to those found for granulocyte O2- production. Thus, the granulocyte O2- generating enzyme system is located on a fraction enriched for plasma membrane markers, and the kinetics of granulocyte production are directly related to the rate and amount of activation of this enzyme.

Functional activity of enucleated human polymorphonuclear leukocytes.

“…It is highly probable, therefore, that the oxidase system is localized in the plasma membrane and uses cytoplasmic substrate(s) for its activity. This conclusion agrees with observations by other investigators (24,26,(28)(29)(30) but contradicts reports on a possible granular component of the oxidase system (31)(32)(33). Probably, upon disruption of the plasma membrane, the molecular configuration of the oxidase system is destroyed or a soluble cytoplasmic factor is lost.…”

“…As far as we know, this is the first description of a subcellular preparation of PMN with a fully intact oxidase osmotic resistance ,°ol system. So-called podosomes have to be prepared from stimulated PMN to show NADPH-oxidase activity; podosomes made from resting PMN cannot be activated (28). Probably, the activation mechanism of the oxidase is destroyed in these particles.…”

The enzyme responsible for the respiratory burst in elicited guinea pig peritoneal macrophages

“…It has been shown in our laboratory (Rossi and Zatti, 1964;Zatti and Rossi, 1965;Rossi and Zatti, 1966;Patriarca et al, 1971;Rossi, Romeo and Patriarca, 1972;Romeo et al, 19733;Patriarca et al, 1975;Rossi, Zabucchi and Romeo, 1975;Bellavite et al, 1980) and other laboratories (Iyer et al;Selvaray and Sbarra, 1967;Paul et al, 1972;Babior Curnutte and Kipnes, 1975;Curnutte, Kipnes and Babior, 1975;De Chatelet et al, 1975;Hohn and Lehrer, 1975;Babior, Curnutte and McMurich, 1976;Iverson et al, 1977;Cohen, Chovaniec and Davies, 1980;Kakinuma and Kaneda, 1980), that in granulocytes and some types of macrophages the enzyme responsible for the respiratory burst preferentially uses NADPH as substrate and is measurable in the particulate fractions of cell homogenates. Other authors postulate that a soluble NADH-oxidase would be the key enzyme which induces the activation of the oxidative metabolism, at least in guinea-pig granulocytes (Evans and Karnovsky, 196 1;Karnovsky, 1962;Baehner, Gilman and Karnovsky, 1970;Karnovsky, 1973;Badwey, Curnutte and Karnovsky, 1979).…”

“…The subcellular localisation of the enzyme responsible for the respiratory burst is controversial. Evidence has been presented showing, in stimulated granulocytes, a localisation in plasma membrane (Briggs et al, 1975;Goldstein et a!., 1975;Segal and Peters, 1977;Dewald et al, 1979;Cohen et al;Rossi et al, 1980) or an association with intracellular organelles (Iverson et al, 1978;Tauber and Goetzl, 1979). According to Andrew et al, (1978) in rabbit alveolar macrophages the oxidase is located in the endoplasmic reticulum.…”

Plasma membrane and phagosome localisation of the activated NADPH oxidase in elicited peritoneal macrophages of the guinea‐pig

“…I N the previous paper (Berton et al, 1982) it has been shown that the stimulation of the respiratory metabolism in peritoneal-elicited macrophages of the guineapig is due to the activation of an oxidase that uses NADPH as substrate and reduces oxygen with the formation of 0;. The kinetic properties of this enzyme are similar to those of the oxidase described in human and guinea-pig granulocytes (Babior, Curnutte and McMurrich, 1976;Cohen. Chovaniec and Davies, 1980;Kakinuma and Kaneda, 1980) and in guinea-pig peritonealresident macrophages (Bellavite et al, 1980).…”