Estimating the initial reaction velocity of a soluble dehydrogenase in situ

The initial reaction velocities (vi) of lactate dehydrogenase in single hepatocytes were determined, by microdensitometry or computer-assisted image analysis, in sections of unfixed mouse liver incubated at 37 degrees C on substrate-containing agarose gel films. They were found to fit the equations vi = 2.82 degrees A and vi = vi + 2 degrees A, where vi and degrees A are, respectively, the 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 microns. The observed and calculated values of vi agreed within 11.5% (n = 71). The validity of the equations for vi was confirmed by showing that the calculated vi was proportional to the thickness of the section and hence the amount of enzyme present. Thus, vi can be determined from measurements of either degrees A alone or vi and degrees A.