No abstract

Specific enzymes play key roles in many pathophysiological processes and therefore are targets for therapeutic strategies. The activity of most enzymes is largely determined by many factors at the post-translational level. Therefore, it is essential to study the activity of target enzymes in living cells and tissues in a quantitative manner in relation to pathophysiological processes to understand its relevance and the potential impact of its targeting by drugs. Proteases, in particular, are crucial in every aspect of life and death of an organism and are therefore important targets. Enzyme activity in living cells can be studied with various tools. These can be endogenous fluorescent metabolites or synthetic chromogenic or fluorogenic substrates. The use of endogenous metabolites is rather limited and nonspecific because they are involved in many biological processes, but novel chromogenic and fluorogenic substrates have been developed to monitor activity of enzymes, and particularly proteases, in living cells and tissues. This review discusses these substrates and the methods in which they are applied, as well as their advantages and disadvantages for metabolic mapping in living cells.

S U M M A R Y CD26/DPPIV is a cell surface glycoprotein that functions both in signal transduction and as a proteolytic enzyme, dipeptidyl peptidase IV (DPPIV). To investigate how two separate functions of one molecule are regulated, we analyzed CD26 protein expression and DPPIV enzyme activity on living human T-helper 1 (Th1) and Th2 cells that express different levels of CD26/DPPIV. DPPIV activity was specifically determined with the synthetic fluorogenic substrate ala-pro-cresyl violet and CD26 protein expression was demonstrated with an FITC-conjugated CD26-specific antibody. Fluorescence of liberated cresyl violet (red) and FITC (green) was detected simultaneously on living T-cells using flow cytometry and spectrofluorometry. Th1 cells expressed three-to sixfold more CD26 protein than Th2 cells. The signal transduction function of the CD26/DPPIV complex, tested by measuring its co-stimulatory potential for proliferation, was directly related to the amount of CD26 protein at the cell surface. However, DPPIV activity was similar in both cell populations at physiological substrate concentrations because of differences in K m and V max values of DPPIV on Th1 and Th2 cells. Western blotting and zymography of Th1 and Th2 wholecell lysates demonstrated similar patterns. This study shows that two functions of one molecule can be controlled differentially.