Biotinylated trypsin and its application as a sensitive, versatile probe for the detection and characterisation of an ovine chondrocyte serine proteinase inhibitor using Western blotting.
Biotinylated trypsin (bT) was used as a probe on Western blots of 10-20% polyacrylamide gradient Tris-Tricine gels for the detection of serine proteinase inhibitors (SPIs) isolated from extracts of ovine articular cartilage and from chondrocyte conditioned culture medium. The major cartilage SPI, a 58 kDa glycoprotein, was purified by sequential Sephacryl S--300 gel permeation, concanavalin A affinity, anion exchange and Superose 12 fast protein liquid chromatography (FPLC). A low molecular weight SPI of approximately 6 kDa was also detectable in cartilage extracts after prolonged storage at 4 degrees C and following affinity chromatography on immobilised chymotrypsin, suggesting that the 6 kDa inhibitor may have arisen from proteolytic modification of the 58 kDa SPI. Analysis of chondrocyte conditioned culture medium using the bT detection system revealed that the major SPI present was the 6 kDa species although a small amount of the 58 kDa SPI was also detectable. The large molecular weight 'native' cartilage SPI was distinct from ovine alpha1-proteinase inhibitor (alpha1-PI) when examined by native polyacrylamide gel electrophoresis (PAGE) and isoelectric focusing (IEF). The 6 kDa cartilage SPI was indistinguishable from basic pancreatic trypsin inhibitor (BPTI, aprotinin) following sodium dodecyl sulfate (SDS)-PAGE under reduced and nonreduced conditions. Amino terminal sequencing of the 6 kDa cartilage SPI revealed a strong (90%) homology with BPTL.
Raymond Purves Bone and Joint Research Laboratories, The Royal North Shore Hospital of Sydney and The University of Sydney, Australia.
K J
KJ
Rodgers
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Melrose
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Ghosh
D000595:Amino Acid Sequence; D000818:Animals; D001710:Biotin / Q000737:chemistry*; D002358:Cartilage, Articular / Q000166:cytology / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010003:Osteoarthritis / Q000209:etiology; D012680:Sensitivity and Specificity; D015842:Serine Proteinase Inhibitors / Q000032:analysis*; D012756:Sheep; D014357:Trypsin / Q000737:chemistry*
D015842:Serine Proteinase Inhibitors; D001710:Biotin; D014357:Trypsin