Cleavage of type XI collagen fibers by gelatinase and by extracts of osteoarthritic canine cartilage.
Gelatinase (matrix metalloproteinase 2) purified from culture medium of MDCK cells by affinity chromatography on gelatin-sepharose was tested against type XI collagen. The purified enzyme-digested native type XI collagen in solution, and as reconstituted fibers, at 30, 34, and 37 degrees C. Both substrates yielded the same digestion products, as characterized by SDS-polyacrylamide gel electrophoresis, but the soluble collagen was cleaved at a higher rate. The first major product seen was an 87-kDa peptide, which was usually associated with one or two peptides migrating between it and alpha 3(XI). With time, a second group of 3 peptides appeared at 78, 75, and 73 kDa. After continued digestion, a third group of peptides was detected with prominent 69- and 67-kDa peptides and minor peptides at 71, 65, and 62 kDa. In overnight (20 hour) digestions, the 60-kDa digestion product accumulated and most of the larger digestion products could no longer be detected. Minor products at 71, 55, and 50 kDa were also noted in these limited digestions. Under the same conditions, denatured type XI was digested to fragments smaller than 13.5 kDa. The enzyme was inhibited by 1,10-phenanthroline or EDTA. Two purified components of cartilage matrix, type II collagen and proteoglycan subunit, as well as crude cartilage homogenates, were not effective inhibitors of the purified enzyme. Similar activity was extracted from canine articular cartilage, and the activity was much stronger in cartilage from osteoarthritic joints than from control joints.
Smith
G N
GN
Specialized Center of Research in Osteoarthritis, Indiana University School of Medicine, Indianapolis 46202.
Hasty
K A
KA
Yu
L P
LP
Lamberson
K S
KS
Mickler
E A
EA
Brandt
K D
KD
D000818:Animals; D002358:Cartilage, Articular / Q000201:enzymology*; D002417:Cattle; D002460:Cell Line; D003094:Collagen / Q000302:isolation & purification / Q000378:metabolism*; D004285:Dogs; D018093:Gelatinases; D007700:Kinetics; D008970:Molecular Weight; D010003:Osteoarthritis / Q000201:enzymology*; D010434:Pepsin A / Q000302:isolation & purification / Q000378:metabolism*; D010446:Peptide Fragments / Q000302:isolation & purification; D013379:Substrate Specificity
D010446:Peptide Fragments; D003094:Collagen; D010434:Pepsin A; D018093:Gelatinases