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Two NADP-cleaving enzymes, namely NADP glycohydrolase and NADP pyrophosphatase, are present in a rat liver extract that inactivates G6PD (glucose 6-phosphate dehydrogenase). The following results suggest that a third G6PD-inactivating protein is present in this extract. (1) Nicotinamide, which selectively inhibits NADP glycohydrolase, enhances the G6PD inactivation under conditions where G6PD activity in control experiments is rather stable. (2) DEAE-cellulose adsorbs the bulk of both NADP glycohydrolase and NADP pyrophosphatase, whereas most of the G6PD-inactivating ability is unadsorbed. (3) Out of 37 liver extracts that were prepared, two were found to lack NADP pyrophosphatase. After removal of NADP glycohydrolase from these extracts by centrifugation, they were still found to inactivate G6PD. (4) Deproteinization of DEAE-cellulose supernatants results in a complete loss of G6PD-inactivating ability; moreover, kinetic experiments performed with the extracts lacking pyrophosphatase strongly support the view that the inactivating protein is an enzyme, although its mechanism is not clear. (5) NADP protects G6PD from inactivation and also reactivates the enzyme completely, thus supporting the view of some action of the inactivating protein on the G6PD-bound NADP.