White blood cells such as granulocytes form an integral part of human host defense against the severe infections that threaten the lives of patients with hematologic disorders and malignancies. An essential part of their function is the generation of superoxide by NADPH oxidase. From the premise that this NADPH oxidase consists of a multicomponent electron transport chain, these studies will examine its development and assembly during both myeloid differentiation and activation of the respiratory burst of phagocytosis. The studies will, in turn, critically evaluate this premise. The induced differentiation of the human, promyelocytic leukemia cell line HL-60 provides the modeI system in which to study the development of the NADPH oxidase during myeloid maturation. The first objective will be to quantitate the development and determine the subcellular localization of individual oxidase components, including the flavoprotein, cytochrome b, and quinone moieties. Resting and stimulated HL-60 cells at various stages of differentiation will be disrupted and fractionated on Percoll gradients. The fractions will be examined for quantitative measurement of each component. Particular attention will be paid to changes in the localization of the NADPH oxidase components during both differentiation and activation to determine whether translocation occurs between subcellular compartments. Studies utilizing anaerobiosis and metabolic inhibitors to stop the flow of electrons at various points along the transport chain will determine the sequence of electron flow in the enzyme complex. Studies of the relationship between the NADPH oxidase and its substrate will determine intracellular levels of NADPH and related purine nucleotides, the ability to produce NADPH by hexosemonophosphate shunt activity, and the affinity of the oxidase for NADPH in developing HL-60 cells. Investigation of the triggering mechanism for activation will focus on the quantity and distribution of changes in membrane potential upon stimulation of cells at various stages of differentiation. Moving from the HL-60 model system, key studies will be repeated in fractionated, normal human bone marrow to corroborate the findings and to delineate any differences between normal and leukemic development of the oxidase system. Chronic granulomatous disease and G6PD-deficient granulocytes will also be examined for possible correlations of NADPH oxidase component defects with cellular superoxide production and clinical disease. (MI)
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