title: Purification and characterisation of 6 and 58 kDa forms of the endogenous serine proteinase inhibitory proteins of ovine articular cartilage.
abstract: The major ovine articular cartilage (AC) serine proteinase inhibitory protein (SPI), a 58 kDa glycoprotein (SPI-58), was purified to homogeneity by sequential Sephacryl S-300 gel permeation, concanavalin A affinity, Mono Q anion exchange and Superose 12 FPLC. If precautions to prevent degradation of the native 58 kDa SPI were not undertaken during the early stages of its purification a SPI of approximately 6 kDa (SPI-6) was generated. SPI-6 could also be generated from SPI-58 by chymotrypsin affinity chromatography, suggesting that SPI-6 could be produced from SPI-58 in vivo by proteolytic processing within the tissue. SPI-6 was indistinguishable from the Kunitz inhibitor, bovine pancreatic trypsin inhibitor (BPTI) by SDS-PAGE under both reducing and non reducing conditions and showed a strong homology to BPTI in N-terminal sequence. These data suggest that the BPTI-like 6 kDa SPI constituted the inhibitory domain of the native 58 kDa SPI of ovine AC. Detection of [14C]-lysine-SPI-6 and SPI-58 in the serum free culture medium from ovine chondrocytes cultured in alginate beads in the presence of [14C]-lysine indicated that these SPIs were chondrocyte biosynthetic products. The inhibitory profiles of SPI-58 and SPI-6 differed somewhat suggesting that each may have an independent role in vivo.
authors.affiliation: Raymond Purves Bone and Joint Research Laboratories, Royal North Shore Hospital of Sydney, St. Leonards, NSW, Australia.
authors.forename: K J
authors.initials: KJ
authors.lastname: Rodgers
authors.forename: J
authors.initials: J
authors.lastname: Melrose
authors.forename: P
authors.initials: P
authors.lastname: Ghosh
mesh_terms: D000818:Animals; D002358:Cartilage, Articular / Q000737:chemistry*; D002417:Cattle; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D006801:Humans; D008970:Molecular Weight; D010449:Peptide Mapping; D015842:Serine Proteinase Inhibitors / Q000302:isolation & purification* / Q000494:pharmacology; D012756:Sheep; D013552:Swine
chemical_list: D015842:Serine Proteinase Inhibitors