[20 Functional Evaluation of Nonphagocytic Nadph Oxidases

By Francis J. Miller, Jr. and Kathy K. Griendling

In neutrophils, NADPH-dependent superoxide (O2") formation is catalyzed by a membrane-bound flavoprotein via the following reaction:

It has become apparent that many cell types have an NAD(P)H oxidase activity similar to that of the neutrophil enzyme. Major differences between phagocytic and nonphagocytic oxidase activities include the kinetics of the reaction, the amount and location of the 02'~ produced, the substrate specificity, and the identity of the enzyme subunits (Table I).

Considerations for Measurement of NAD(P)H Oxidase Activity

To accurately assign a role for an NAD(P)H oxidase in the generation of O2'-, oxidase activity must be measured and shown to be inhibited by appropriate pharmacological or molecular agents. This section addresses tissue preparation and special considerations to ensure that the activity is attributable to this enzyme, and the latter half of this chapter deals mainly with accurate measurement of O2 ~ production, a prerequisite for assessment of NAD(P)H oxidase activity.

Preparation ofHomogenate and Membrane Fractions

The nonphagocytic NAD(P)H oxidase is sensitive to methods of homogenate preparation. The enzyme resides in the membrane fraction, and often the signal-to-noise ratio is significantly enhanced by careful preparation of membrane fractions. Rough handling of cells or tissue can dramatically increase baseline 02'~ production, so mild lysis methods are preferable. Care must be taken to minimize disruption of mitochondria to avoid the confounding contribution of mitochondrial NADH-dependent enzymes.

Cultured Cells. After washing with ice-cold phosphate-buffered saline (PBS), cells are scraped from the plate in 5 ml of this same solution, and centrifuged at 750g at 4° for 10 min.1 The supernatant is discarded, and the pellet is re-suspended in lysis buffer containing protease inhibitors. Both a phosphate buffer [20 mM monobasic potassium phosphate (pH 7.4), aprotinin (10 /xg/ml), leupeptin

1 K. K. Griendling, c. A. Minieri, J. D. Ollerenshaw, and R. W. Alexander, Circ. Res. 74,1141 (1994).

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