AN ENZYME-HISTOCHEMICAL INVESTIGATION OF CYSTEINE ENDOPROTEASE ACTIVITY IN CHICKEN BONE TISSUE

Cysteine endoprotease activity was demonstrated in chicken osteoclasts using an azo-dye method. The activity was also observed in cartilage matrix where an active osteoclast attacked, but not in bone matrix. No significant activity was seen in osteoblasts and osteocytes. The enzy me hydrolyzed -phenylalanyl-L-arginine-4-methoxy-2-naphthylamide (Z-Ph e-Arg-MNA), but not Z-Arg-Arg- nor Z-Ala-Arg-Arg-MNA. Furthermore, the activity was dependent on mercaptoethylamine, an SH-reagent. A glutar aldehyde-containing fixative was needed for demonstration of the activ ity. There was no significant activity in 4% paraformaldehyde-fixation samples, suggesting that this soluble enzyme protein might diffuse ou t from the cell during histochemical prodecures in mild fixed sapmles. This is the first report showing the presence of SH-reagent dependent Z-Phe-Arg-MNA hydrolyzing activity in osteoclasts in vivo.