TY - JOUR

T1 - Microfluorometric kinetic analysis of cathepsin B activity in single human thyroid follicular epithelial cells using image analysis and continuous monitoring

AU - Kayser, L.

AU - Perrild, H.

AU - Thomsen, J.

AU - Høyer, P. E.

PY - 1996

Y1 - 1996

N2 - The activity of a cysteine proteinase, cathepsin B (EC 3.4.22.1), was determined in unfixed single human thyroid folIicular epithelial cells at room temperature using an image analysis system. The formation of the reaction product was monitored every minute by measuring the increasing fluorescence intensity of emitted light from a Schiff-base product formed by the substrate N-CBZ-ala-arg-arg-4-methoxy-2-naphthylamide and the coupling agent 5-nitrosalicylaldehyde. Non-specific fluorescent 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 increased 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 another linear increase for 11 min (range 8-14 min). The second linear part of the curve was not as steep as the first one. The reaction velocity recorded in individual granules resulted either in a biphasic curve or straight line, suggesting the presence of two distinct organelle compartments with differences in membrane permeability. It is concluded that human thyroid follicular epithelial cells in culture exhibit cathepsin B activity which can be monitored continuously by videomicroflurometry without interference from non-specific fluorescence.

AB - The activity of a cysteine proteinase, cathepsin B (EC 3.4.22.1), was determined in unfixed single human thyroid folIicular epithelial cells at room temperature using an image analysis system. The formation of the reaction product was monitored every minute by measuring the increasing fluorescence intensity of emitted light from a Schiff-base product formed by the substrate N-CBZ-ala-arg-arg-4-methoxy-2-naphthylamide and the coupling agent 5-nitrosalicylaldehyde. Non-specific fluorescent 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 increased 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 another linear increase for 11 min (range 8-14 min). The second linear part of the curve was not as steep as the first one. The reaction velocity recorded in individual granules resulted either in a biphasic curve or straight line, suggesting the presence of two distinct organelle compartments with differences in membrane permeability. It is concluded that human thyroid follicular epithelial cells in culture exhibit cathepsin B activity which can be monitored continuously by videomicroflurometry without interference from non-specific fluorescence.

UR - http://www.scopus.com/inward/record.url?scp=0029941057&partnerID=8YFLogxK

U2 - 10.1007/BF02409013

DO - 10.1007/BF02409013

M3 - Journal article

C2 - 8762057

AN - SCOPUS:0029941057

VL - 28

SP - 257

EP - 263

JO - The Histochemical Journal

JF - The Histochemical Journal

SN - 0018-2214

IS - 4

ER -