Enzyme Cytochemical Techniques for Metabolic Mapping in Living Cells, with Special Reference to Proteolysis

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.

Fluorescent labeling of tRNAs for dynamics experiments

“…Labeling stoichiometries were determined from the ratio of absorption in the visible (462 nm for proflavin, 512 nm for rhodamine 110) to that at 260 nm for tRNA (corrected for a contribution from the dye). The lower stoichiometry in the case of rhodamine may be due to its expected lower nucleophilicity as compared with proflavin, given its much lower pK a value (rhodamine 110, 4.3 [Boonacker and Van Noorden, 2001]; proflavin, 9.6 [Horobin et al 2006]) and its more hindered primary amine.…”

Impacts of sarA and agr in Staphylococcus aureus Strain Newman on Fibronectin-Binding Protein A Gene Expression and Fibronectin Adherence Capacity In Vitro and in Experimental Infective Endocarditis

“…To quantify overall protease activity, a microplate assay kit (Molecular Probes, Eugene, Oreg.) was utilized as previously described (4). Protease activ-ity was slightly decreased in the agr mutant (ϳ0.8-fold) but significantly increased in the sarA mutant (three-to fivefold; P Ͻ 0.05) compared to the parental strain (Fig.…”

Fluorogenic Substrate [Ala-Pro]2-Cresyl Violet But Not Ala-Pro-Rhodamine 110 Is Cleaved Specifically by DPPIV Activity: A Study in Living Jurkat Cells and CD26/DPPIV-transfected Jurkat Cells

“…Excitation for cresyl violet was performed at 591 nm with a slit width of 10 nm and emission was detected at 628 nm with a slit width of 10 nm (Boonacker and Van Noorden 2001). Rhodamine 110 was excited at 491 nm with a slit width of 10 nm and emission was detected at 529 nm with a slit width of 10 nm (Boonacker and Van Noorden 2001). Fluorescence was measured continuously during the first 4 min of incubation.…”