Analytical Subcellular Fractionation of Human Granulocytes with Special Reference to the Localization of Enzymes Involved in Microbicidal Mechanisms

Abstract

1. Granulocytes were isolated from 40–400 ml of human blood, disrupted and subjected to analytical subcellular fractionation by centrifugation on sucrose density gradients. 2. The principal subcellular organelles, characterized by their respective marker enzymes, were resolved on the density gradients. The subcellular localization of hitherto unassigned enzymes was determined by comparison with the marker enzymes. 3. NAD(P)H-dependent reduction of nitroblue tetrazolium at high nucleotide concentration (2·5 mmol/l) and NAD(P)H/cytochrome c reduction has a complex distribution with activity in the plasma membrane, mitochondria and cytosol fractions. 4. At a lower, physiological concentration of pyridine nucleotides (25 μmol/l), NADH-dependent nitroblue tetrazolium reduction was confined to the plasma membrane whereas NADPH dependent-reduction of the dye was undetectable. NAD(P)H/nitroblue tetrazolium reductase activity was partially inhibited by superoxide dismutase. 5. NADPH and NADH oxidase activities, measured by the production of oxidized nucleotide, were localized with the azurophil granules. 6. Superoxide dismutase, catalase, glutathione reductase and glutathione peroxidase were localized predominantly to the cytosol. 7. Neutral α-glucosidase and γ-glutamyl transpeptidase showed a bimodal distribution with localizations to both endoplasmic reticulum and specific granule components. 8. It is suggested that the plasma membrane, and thus the wall of phagocyte vacuoles, contains a free radical generating NADH-dependent enzyme which plays a primary bactericidal role. The cytosol contains mechanisms which detoxify hydrogen peroxide, superoxide and free radicals and protect the granulocyte from these compounds.