Monitoring a Heterogeneous Coupled System Using Soluble and Biologically Localized Firefly Luciferase as a Probe for Bulk and Local [ATP]

Abstract

Classical biochemistry has provided a great deal of information about isolated catalytic units (from enzymes to organelles), by studying their properties in dilute solution or suspension. The results are often directly projected on their functionality in vivo, implicitly assuming a homogeneous behavior in situ. However, a critical look at the cellular environments^1,2 strongly refutes the latter assumption and indicates that special kinetic treatments would rather be required to describe these situations, taking into account physical steps (diffusion, partition, etc.) inherent to heterogeneous systems, in addition to the intrinsic properties of the catalysts as observed in homogeneous phase³. Such treatment has been successfully applied for immobilized enzyme systems⁴, stressing that the activity of the enzymes is exclusively determined by the local concentration of reactants in their own microenvironment. However, in these systems only the bulk concentrations of reactants have been assessed. It is thus important to have means to monitor also local events which reflect more directly the activity of enzymes in situ.