MICROFLUOROMETRIC KINETIC-ANALYSIS OF CATHEPSIN-B ACTIVITY IN SINGLE HUMAN THYROID FOLLICULAR EPITHELIAL-CELLS USING IMAGE-ANALYSIS AND CONTINUOUS MONITORING

The activity of a cysteine proteinase, cathepsin B (EC 3.4.22.1), was determined in unfixed single human thyroid follicular epithelial cells at room temperature using an image analysis system. The formation of the reaction product was monitored every minute by measuring the incre asing fluorescence intensity of emitted light from a Schiff-base produ ct formed by the substrate N-CBZ-ala-arg-arg-4-methoxy-2-naphthylamide and the coupling agent 5-nitrosalicylaldehyde. Non-specific fluoresce nt signals were eliminated by adjusting the video signal to zero using leupeptin, a specific inhibitor of cathepsins B, H and L. The enzyme activity was expressed as fluorescence intensity versus time. After a mean lag period of 5 min (range 4-8 min, n=4), the enzyme activity inc reased linearly lasting on average 5 min (range 3-8 min), followed by a marked decrease in reaction rate for 3 min (range 1-4 min), and anot her linear increase for 11 min (range 8-14 min). The second linear par t of the curve was not as steep as the first one. The reaction velocit y recorded in individual granules resulted either in a biphasic curve or straight line, suggesting the presence of two distinct organelle co mpartments with differences in membrane permeability.It is concluded t hat human thyroid follicular epithelial cells in culture exhibit cathe psin B activity which can be monitored continuously by videomicrofluor ometry without interference from non-specific fluorescence.