Analysis of enzyme reactions in situ.

Estimations of metabolic rates in cells and tissues and their regulation on the basis of kinetic properties of enzymes in diluted solutions may not be applicable to intact living cells or tissues. Enzymes often behave differently in living cells because of the high cellular protein content that can lead to homologous and heterologous associations of protein molecules. These associations often change the kinetics of enzymes as part of post-translational regulation mechanisms. An overview is given of these interactions between enzyme molecules or between enzyme molecules and structural elements in the cell, such as the cytoskeleton. Biochemical and histochemical methods are discussed that have been developed for in vivo and in situ analyses of enzyme reactions, particularly for the study of effects of molecular interactions. Quantitative (histochemical) analysis of local enzyme reactions or fluxes of metabolites has become increasingly important. At present, it is possible to calculate local concentrations of substrates in cells or tissue compartments and to express local kinetic parameters in units that are directly comparable with those obtained by biochemical assays of enzymes in suspensions. In situ analysis of the activities of a number of enzymes have revealed variations in their kinetic properties (Km and Vmax) in different tissue compartments. This stresses the importance of in vivo or in situ analyses of cellular metabolism. Finally, histochemical determinations of enzyme activity in parallel with immunohistochemistry for the detection of the total number of enzyme molecules and in situ hybridization of its messenger RNA allow the analysis of regulation mechanisms at all levels between transcription of the gene and post-translational activity modulation.