Pette D: Microphotometric determinations of enzyme activity in single cells in cryostat sections. II. Succinate dehydrogenase, lactate dehydrogenase arid triosephosphate dehydrogenase activities in red, intermediate and white fibers of soleus and rectus f

A gel film technique was adopted for kinetic enzyme activity determination in single cells using cryostat sections by means of a microscope photometer. The main principle of the method is a comparative activity determination, based on biposi-tional recording of initial reaction kinetics in two preselected measuring fields in the same tissue section. Systematic model experiments were performed to prove the fulfillment of the following conditions: (a) validity of Beer’s law; (b) optimal con-centrations of substrates, cosubstrates and dye within the reaction film, including evidence that the reaction rate is not limited by insufficient diffusion of these com-pounds; (c) proportionality between local enzyme concentration or thickness of tissue sections and the recorded reaction rate; and (d) specificity of the method as demonstrated by studying reaction rates in the absence of the substrate as well as in the presence of substrate and a specific inhibitor. The validity of the method was also examined by comparing the levels of succinate dehydrogenase activity in vari-ous rat tissues, as measured microphotometrically, with the enzyme levels as deter-mined in homogenates. Microphotometric assays for nicotinamide adenine dints