Kinetic analysis of lactate dehydrogenase in situ in mouse liver determined with a quantitative histochemical technique

Summary The kinetics of lactate dehydrogenase in situ were studied in sections of unfixed liver of the male mouse using a quantitative histochemical technique. The sections were incubated on substrate-containing gel films. The absorbance of the final reaction products deposited in a single hepatocyte was measured continuously during the incubation as a function of incubation time using a scanning microdensitometer. The absorbance increased non-linearly during the first minute of incubation, but linearly for at least the next 3 min afterwards. The initial velocity (v_i) of the dehydrogenase was calculated from two equations proposed previously by us, v_i=2.82 °A and v_i=v+2°A, where v and °A are, respectively, the gradient and intercept o linear regression line of absorbance on time for incubation times between 1 and 3 min.The dependence of v_i on lactate concentration gave the following mean kinetic constants. For periportal hepatocytes, the apparent K_m=14 mM and V_max=80 moles hydrogen equivalents formed cm^–3 hepatocyte cytoplasm min^–1. For pericentral hepatocytes, K_m=12 mM and V_max=87 moles hydrogen equivalents cm^–3 min^–1. The K_m values are very similar to those determined previously from biochemical assays. The concentrations of the enzyme in single hepatocytes calculated from the V_max values are in good agreement with those obtained by another method. These data substantiate the validity of our equations.