Figure 6. Calculation of kinetic parameters for HPETE-dependent NO consumption by PGHS-2.

(A) K_m (app) was determined by measuring initial rates of NO consumption by 9.6 nM PGHS-2 at various 15(S)-HPETE concentrations. Michaelis–Menten parameters were calculated using Enzfitter (Elsevier–Biosoft). Rates of NO consumption are plotted as v against v/[S]. (B) K_m (app) was determined as described in (A) but by varying the NO concentration. (C) HPETE (5 μM) was incubated for 5 min with or without 9.6 nM PGHS-2 and 7.2 μM NO in Tris buffer, pH 7.8, before being extracted using acidified diethyl ether, as described in the Experimental section, and reconstituted in methanol. Samples were scanned from 200 to 350 nm using methanol as reference: (a) control, (b) with PGHS-2 plus NO.