THE SERINE PROTEINASE INHIBITORY PROTEINS OF THE CHONDRODYSTROPHOID (BEAGLE) AND NON-CHONDRODYSTROPHOID (GREYHOUND) CANINE INTERVERTEBRAL DISC

Trypsin inhibitory proteins of low buoyant density (p less than or equ al to 1.35 g/mL) fractions were prepared by CsCl density gradient ultr acentrifugation of 4 M guanidinium hydrochloride extracts of lumbar be agle and greyhound annulus fibrosus and nucleus pulposus from animals aged 1 to 6 years. Affinity blotting with biotinylated trypsin was use d to identify active trypsin inhibitory protein species; these species were also identified immunologically by Western blotting using antibo dies against bovine pancreatic trypsin inhibitor (BPTI), and human int er-alpha-trypsin inhibitor (ITI). None of the trypsin inhibitory speci es evident in Western blots were reactive with anti-human alpha(1)-pro teinase inhibitor (alpha-(1)-PI), alpha(2)-macroglobulin or secretory leucocyte proteinase inhibitor. The greyhound intervertebral disc samp les generally had higher levels of active trypsin inhibitor species pe r unit weight of tissue extracted, and a more extensive range of inhib itor species. Inhibitor species of 30, 32, 34 kDa were identified in b oth beagle and greyhound intervertebral disc samples; these species we re generally most prominent in the annulus fibrosus samples. In contra st, the nucleus pulposus samples contained relatively large trypsin in hibitor species; the anti-BPTI detected an inhibitor species of simila r to 85-90 kDa; anti-ITI detected species of 120-250 kDa; biotinylated trypsin detected species of 60-110 kDa. A small molecular mass trypsi n inhibitor species of 6 kDa, which was of similar mobility to BPTI, w as also detected in annulus fibrosus samples; however, this species di d not react with anti-BPTI.