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  • Title: The kinetics of enzymes in situ, with special reference to lactate and succinate dehydrogenases.
    Author: Nakae Y, Stoward PJ.
    Journal: Histol Histopathol; 1995 Apr; 10(2):463-79. PubMed ID: 7599442.
    Abstract:
    The kinetics of two enzyme systems in situ that have been studied with real-time image analysis systems are reviewed in detail. The enzymes are a structurally-bound mitochondrial enzyme, succinate dehydrogenase (SDH) and a soluble cytoplasmic enzyme, lactate dehydrogenase (LDH). The image analysis system is used to capture successive images of a tissue section at constant time intervals whilst it is being incubated on a substrate-containing gel film. The increasing absorbances of the final reaction products in each cell are measured in the successive images as a function of incubation time. The absorbances of the formazan reaction products formed by SDH, for example, in sections of liver determined by such means increase linearly during the first minute of incubation, but non-linearly afterwards. The initial velocities of SDH in single hepatocytes in sections incubated on gel substrate films are calculated from the activities during the first 20 s of incubation. In contrast, the activities of LDH measured in various cell types, including hepatocytes, with the gel film technique increase nonlinearly during the first minute of incubation, but linearly for incubation times between 1 and 3 min. The initial velocities (vi) of LDH in single cells can be, calculated, however from the activities during the first interval, 10 s, of the image capturing sequence. Unfortunately, the experimental errors of the initial velocities of LDH determined in this way are relatively high. To overcome this problem, we have found empirically that the equations vi = a1oA and vi = v + a2oA enable reliable initial velocities (vi) of the LDH reaction in single cells of various types to be calculated using the data of the linear activities for incubation times between 1 and 3 min. Dependence of the initial velocities of the SDH and LDH reactions on substrate concentrations gave the Michaelis constants (Km) and maximum velocities (Vmax). The Km values determined in situ for SDH in hepatocytes and for LDH in various cell types with the gel film technique are in the same order of magnitude as the corresponding values determined biochemically. The constants a1, a2 and Km of LDH are characteristic for each cell type and seem to be related to the intracellular localization of the enzyme and to its ligand-binding rather than to the different isozyme compositions in various cell types.
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