The overall objective of this proposal is an investigation of the oxidative metabolism of the human neutrophil with particular emphasis on the relationship of this metabolism to the bactericidal activity of the cell. We are especially interested in defining the metabolic basis of the respiratory burst and the factors which are responsible for the expression of this actvity during phagocytosis. The following specific aims are directed toward this goal: 1. Examination of the kinetics of NAD(P)H oxidation using a semi-purified granule fraction obtained from sucrose density gradient fractionation. Both normal and CGD cells will be employed under both resting and phagocytizing conditions. 2. Attempt to delineate the mechanism whereby the oxidase apparently switches from allosteric to hyperbolic kinetics. This will involve use of possible model systems (e.g., cyclic nucleotides and sulfhydryl compounds) as well as a search for endogenous allosteric modifiers in various fractions of the sucrose gradient. 3. Continue efforts to purify the oxidase(s) employing the gradient isolation (which results in a thirty to fifty fold increase in specific activity) as a first step. We shall first attempt to solubilize the enzyme(s) by the use of detergents, high ionic strength, butanol extraction, etc. The solubilized enzyme(s) can then be further purified by conventional techniques. Such enzyme purification will allow us to unequivocally determine whether the cell possesses two separate oxidases or a single enzyme with dual specificity towards either NADH or NADPH. It will also permit kinetic determinations which are more meaningful than those done in a crude system containing possible competing reactions.