Kinetic Parameters of Lactate Dehydrogenase in Liver and Gastrocnemius Determined by Three Quantitative Histochemical Methods

We determined the Michaelis constant (Km) and maximal velocity (Vmax) of lactate dehydrogenase (LDH) in periportal hepatocytes and skeletal muscle fibers by three different histochemical assay methods. Unfixed sections of mouse liver and gastrocnemius were incubated at 37C either on substrate (l-lactate)-containing agarose gel films or in aqueous assay media with and without 18% polyvinyl alcohol (PVA) as a tissue protectant. The absorbances of the formazan final reaction products were continuously measured at 584 nm in the cytoplasm of individual cells as a function of incubation time, using an image analysis system. The kinetic parameters of purified rabbit skeletal muscle LDH incorporated into polyacrylamide gel sections were similarly determined. The intrinsic initial velocities (vi) of LDH, corrected for “nothing dehydrogenase,” were determined as described in the previous article. The Km and Vmax were calculated from Hanes plots of s/vi on l-lactate concentration (s). The Km values obtained with thr...