Dynamic measurements of intracellular aminopeptidase activity in hepatocytes using multiparameter digitized video fluorescent microscopy

Digitized video fluorescent microscopy (DVFM) is a powerful technique for quantitating multiple processes in living cells. However, techniques for measuring protease activity by DVFM are not available. Our aim was to develop an approach for measuring aminopeptidase activity using DVFM. We conjugated glycine-7-amino-4-methylcoumarin-3-acetic acid (glycine-AMC-3-acetic acid) to dextran using a PEG bridge. Glycine-AMC-3-acetic acid-PEG-dextran was microinjected into cultured rat hepatocytes along with rhodamine-dextran. Glycine-AMC-3-acetic acid-PEG-dextran is nonfluorescent, but aminopeptidase hydrolysis of the glycine-AMC bond liberates the fluorescent AMC-3-acetic acid-PEG-dextran within the cell. Following microinjection, rhodamine-dextran fluorescence remained constant while AMC-3-acetic acid-PEG-dextran fluorescence increased in a linear fashion over time reflecting proteolytic cleavage of the glycine-AMC bond. AMC-3-acetic acid-PEG-dextran and rhodamine-dextran fluorescence were cytosolic as evidenced by diffuse fluorescence and colocalized. Because rhodamine-dextran fluorescence remained constant and the probes colocalized, the fluorescent ratio of AMC-3-acetic acid-PEG-dextran/rhodamine-dextran could be used to measure proteolysis. Basal rates of proteolysis were 9 ± 3 ratio units/10 min Comicroinjection of the aminopeptidase inhibitor, bestatin, along with the dextran probes abolished proteolysis. Addition of the calcium ionophore, 4-Br-A23187, increased proteolysis 12-fold to 107 ± 14/10 min (P <0.01). We have developed a novel, dynamic technique for measuring pH-sensitive, Ca²⁺-dependent aminopeptidase activity in living cells using DVFM. This approach may be used for the measurement of other peptidase activities bysynthesizing peptidase-specific peptidyl-AMC-3-acetic acid-PEG-dextran conjugates.