Fluorescence microscopic demonstration of cathepsin K activity as the major lysosomal cysteine proteinase in osteoclasts.
The enzyme activity of lysosomal cysteine proteinases in vital rabbit osteoclasts and mouse osteoclast-like cells was visualized with Z-Leu-Arg-4-methoxy-beta-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 A1 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.
Kamiya
T
T
Department of Oral and Maxillofacial Surgery, Nagasaki University School of Dentistry, 1-7-1 Sakamoto, Nagasaki 852-8588.
Kobayashi
Y
Y
Kanaoka
K
K
Nakashima
T
T
Kato
Y
Y
Mizuno
A
A
Sakai
H
H
D000595:Amino Acid Sequence; D000818:Animals; D056657:Cathepsin K; D002403:Cathepsins / Q000378:metabolism*; D002478:Cells, Cultured; D005260:Female; D008247:Lysosomes / Q000201:enzymology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008856:Microscopy, Fluorescence; D010010:Osteoclasts / Q000201:enzymology*; D011817:Rabbits
D002403:Cathepsins; D056657:Cathepsin K; C534132:Ctsk protein, mouse