Initial reaction kinetics of succinate dehydrogenase in mouse liver studied with a real-time image analyser system

Summary The initial reaction kinetics of succinate dehydrogenase in situ were investigated in sections of mouse unfixed liver using an ARGUS-100 image analyser system. The sections were incubated on substrate-containing agarose gel films. Images of a section, illuminated with monochromatic light (584 nm), were captured with the image analyser in real time at intervals of 10 s during the incubation. The absorbances of selected hepatocytes in the successive images were determined as a function of time. In every cell, the absorbance increased non-linearly after the first minute of incubation. The initial velocity of the dehydrogenase was calculated from the linear activities during the first 20 s of incubation. Hanes plots of the initial velocities and succinate concentration yielded the following mean kinetic constants. For periportal hepatocytes, the apparentK _m=1.2±0.8 mM andV _max=29±2 mol hydrogen equivalents formed/cm³ hepatocyte cytoplasm per min. For pericentral hepatocytes,K _m=1.4±1.0 mM andV _max=21±2 mol hydrogen equivalents/cm³ per min. TheK _m values are very similar to those determined previously from biochemical assays. These results, and the observed dependence of the initial velocity on the enzyme concentration, suggest that the technique reported here is valid for the histochemical assay of succinate dehydrogenase.