MICROPHOTOMETRIC DETERMINATION OF ENZYME ACTIVITY IN SINGLE CELLS IN CRYOSTAT SECTIONS I. APPLICATION OF THE GEL FILM TECHNIQUE TO MICROPHOTOMETRY AND STUDIES ON THE INTRALOBULAR DISTRIBUTION OF SUCCINATE DEHYDROGENASE AND LACTATE DEHYDROGENASE ACTIVITIES IN RAT LIVER

Abstract

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 bipositional 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 concentrations of substrates, cosubstrates and dye within the reaction film, including evidence that the reaction rate is not limited by insufficient diffusion of these compounds; ( 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 various rat tissues, as measured microphotometrically, with the enzyme levels as determined in homogenates. Microphotometric assays for nicotinamide adenine dinucleotide phosphate isocitrate dehydrogenase, lactate dehydrogenase and succinate dehydrogenase are described. Using these techniques it was found that: the periportal and central areas of the hepatic lobule in rat contain the same level of lactate dehydrogenase activity; succinate dehydrogenase activity is 1.6 times higher in the periportal area as compared to the centrilobular area. In experimental thyrotoxicosis, the ratio of enzyme activities in the periportal and central areas was 1.1.