Detection of femtomole quantities of mature cathepsin K with zymography.
Cathepsin K, the most potent mammalian collagenase, has been implicated in osteoporosis, cancer metastasis, atherosclerosis, and arthritis. Although procathepsin K is stable and readily detected, the active mature cathepsin K eludes detection by in vitro methods due to its shorter half-life and inactivation at neutral pH. We describe, for the first time, reliable detection, visualization, and quantification of mature cathepsin K to femtomole resolution using gelatin zymography. The specificity of the method was validated with cathepsin K knockdown using small interfering RNA (siRNA) transfection of human monocyte-derived macrophages, and enzymatic activity confirmed with benzyloxycarbonyl-glycine-proline-arginine-7-amino-4-methylcoumarin (Z-GPR-AMC) substrate hydrolysis was fit to a computational model of enzyme kinetics. Furthermore, cathepsin K zymography was used to show that murine osteoclasts secrete more cathepsin K than is stored intracellularly, and this was opposite to the behavior of the macrophages from which they were differentiated. In summary, this inexpensive, species-independent, antibody-free protocol describes a sensitive method with broad potential to elucidate previously undetectable cathepsin K activity.
Li
Weiwei A
WA
Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30332, USA.
Barry
Zachary T
ZT
Cohen
Joshua D
JD
Wilder
Catera L
CL
Deeds
Rebecca J
RJ
Keegan
Philip M
PM
Platt
Manu O
MO
D000818:Animals; D056657:Cathepsin K / Q000032:analysis* / Q000235:genetics; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005780:Gelatin / Q000737:chemistry; D055785:Gene Knockdown Techniques; D006801:Humans; D007700:Kinetics; D008264:Macrophages / Q000201:enzymology / Q000276:immunology; D051379:Mice; D034741:RNA, Small Interfering / Q000378:metabolism
D034741:RNA, Small Interfering; D005780:Gelatin; D056657:Cathepsin K