Estimating the initial reaction velocity of a soluble dehydrogenase in situ

Summary The initial reaction velocities (v_i) of lactate dehydrogenase in single hepatocytes were determined, by microdensitometry or computer-assisted image analysis, in sections of unfixed mouse liver incubated at 37°C on substrate-containing agarose gel films. They were found to fit the equations v_i=2.82°A and v_i=v+2°A, where v and °A are, respectively, gradients (or steady-state linear velocities) and the intercepts on the absorbance axis of the linear regression lines of the absorbance (A) on incubation time plots for incubation times between 1 and 3 min. Both equations were independent of section thickness between 4 and 14 m. The observed and calculated values of v_i, agreed within 11.5% (n = 71). The validity of the equations for v_i was confirmed by showing that the calculated v_i was proportional to the thickness of the section and hence the amount of enzyme present. Thus, v_i can be determined from measurements of either °A alone or v and °A.