Immunohistochemical and biochemical demonstration of calcium-dependent cysteine proteinase (calpain) in calcifying cartilage of rats.
Calpain is a Ca2(+)-dependent cysteine proteinase that has neutral pH optima. There are two classes of calpains that differ in their optimal calcium ion concentration for enzymatic activity. Calpain I requires a low concentration of Ca2+ for activation, and calpain II requires a much higher Ca2+ concentration. This report describes the immunohistochemical and biochemical demonstration of calpain II in calcifying cartilage in rats and also the degradation of the cartilage proteoglycan subunit by calpain II. Immunoperoxidase (peroxidase-antiperoxidase) staining of the frozen sections of the knee joint from 3-day-old and 6-day-old Wistar rats, using polyclonal antibodies against the respective heavy subunits of calpains I and II, showed positive staining only with the anti-calpain II antibody in the hypertrophic chondrocytes and surrounding cartilaginous matrix of the growth cartilage. Diethylaminoethyl-cellulose chromatography of the cartilaginous extract from 3-day-old rats showed a peak of caseinolytic activity attributable to calpain as well as an inhibitory peak of calpastatin, a specific inhibitor protein of calpains. Immunoblotting using the anti-calpain II antibody of the calpain peak demonstrated identity with the heavy subunit of calpain II (80 kDa). Proteoglycan-degrading activity of calpain was assessed using porcine kidney calpain II and the porcine articular cartilage proteoglycan subunit. After incubation in the presence of Ca2+, degradation of proteoglycan was demonstrated by the change of the elution position on Sepharose-2B chromatography. It is possible that calpain functions as one of the proteoglycan-degrading proteolytic enzymes of growth cartilage. Intracellular localization of calpain in hypertrophic chondrocytes also suggests a role in the hypertrophic process of the chondrocyte in growth cartilage.
Shimizu
K
K
Department of Orthopaedic Surgery, Faculty of Medicine, Kyoto University, Japan.
Hamamoto
T
T
Hamakubo
T
T
Lee
W J
WJ
Suzuki
K
K
Nakagawa
Y
Y
Murachi
T
T
Yamamuro
T
T
D000818:Animals; D002113:Calcification, Physiologic / Q000502:physiology; D002118:Calcium / Q000502:physiology; D002154:Calpain / Q000378:metabolism*; D006132:Growth Plate / Q000166:cytology / Q000378:metabolism* / Q000502:physiology; D015151:Immunoblotting; D007122:Immunoelectrophoresis; D007150:Immunohistochemistry; D007719:Knee Joint / Q000166:cytology / Q000378:metabolism / Q000502:physiology; D011509:Proteoglycans / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains
D011509:Proteoglycans; D002154:Calpain; D002118:Calcium