J. Biochem, 1998, Vol. 123, No. 4 752-759
© 1998 Japanese Biochemical Society
research-article |
Fluorescence Microscopic Demonstration of Cathepsin K Activity as the Major Lysosomal Cysteine Proteinase in Osteoclasts
,1
*Department of Oral and Maxillofacial Surgery, Nagasaki University School of Dentistry 1-7-1 Sakamoto, Nagasaki 852-8588
Department of Orthodontics, Nagasaki University School of Dentistry 1-7-1 Sakamoto, Nagasaki 852-8588
Department of Pharmacology, Nagasaki University School of Dentistry 1-7-1 Sakamoto, Nagasaki 852-8588
Department of Hospital Pharmacy, Nagasaki University School of Medicine Nagasaki 852-8523
1To whom correspondence should be addressed. E-mail: h-sakai{at}net.nagasaki-u.ac.jp
The enzyme activity of lysosomal cysteine proteinases in vital rabbit osteoclasts and mouse osteoclast-like cells was visualized with Z-Leu-Arg-4-methoxy-ß-naphthylamide (Z-LR-MNA) as the enzyme substrate. The MNA liberated by proteolysis forms a fluorescent insoluble Schiff-base product in the presence of 5-nitrosalicylaldehyde. Many small fluorescent particles, endproducts of the Z-LR-MNA hydrolysis, were observed in proximity to the bone surface underneath the actively resorbing osteoclasts, as well as in the cytoplasm. The Z-LR-MNA hydrolase activity was markedly diminished by bafilomycin Al and chloroquine treatment. Moreover, the activity was completely inhibited by cysteine proteinase inhibitors such as leupeptin and E-64d, but not by other classes of proteinase inhibitors. About 60% of the hydrolase activity in mouse osteoclast-like cells was immunoabsorbed by anti-cathepsin K antibody-coupled Sepharose CL-4B beads, and about 10% of the activity was absorbed with the anti-cathepsin L antibody-coupled beads. Thus, the majority of the Z-LR-MNA hydrolase activity in osteoclasts was derived from cathepsin K. In contrast, using the same substrate in the assay, no detectable cathepsin K activity was observed in mouse peritoneal macrophages. The abundant cathepsin K activity in osteoclasts would therefore indicate a significant role of this enzyme in bone matrix degradation.
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