The serine proteinase inhibitory proteins of the human intervertebral disc: their isolation, characterization and variation with ageing and degeneration.
Serine proteinase inhibitory proteins (SPIs) were extracted from human disc tissues using 2 M GuHCl and subjected to CsCl density gradient ultracentrifugation. The SPIs recovered in the low buoyant density fractions (rho < or = 1.35 g/ml) were purified by a combination of gel-permeation, ion-exchange, trypsin affinity, and reverse-phase high performance chromatographies. Characterisation of the major disc SPI by polyacrylamide gel electrophoresis, isoelectric focussing, enzyme inhibition and pH stability studies indicated that this small molecular weight (12-14 kDa), highly basic (pI > 9.5), acid-stable but alkaline-labile protein possessed potent inhibitory activity against bovine pancreatic trypsin and chymotrypsin, and human leukocyte elastase and cathepsin G. Two-major and two-minor low molecular weight cationic SPI species were identified by reverse-phase HPLC. The predominant species was identical to a human articular cartilage SPI sharing amino terminal sequence homology with the mucus proteinase inhibitors (MPIs). It also cross-reacted with an antiserum to the MPIs and behaved identically to secretory leucocyte proteinase inhibitor (SLPI) when examined by reverse phase HPLC, and SDS PAGE. A higher molecular weight (54 kDa), anionic (pI approximately 4.6) SPI was also purified and identified as alpha 1-proteinase inhibitor (alpha 1-PI). Quantification of alpha 1-PI and the small molecular weight cationic disc inhibitors indicated that the latter were depleted in morphologically degenerate disc tissues while levels of alpha 1-PI were somewhat higher although a large proportion of the alpha 1-PI was inactive. A depletion of total SPI levels was evident overall in degenerate discs suggesting a functional role for these inhibitory proteins in the maintenance of IVD matrix homeostasis.
Melrose
J
J
Raymond Purves Research Laboratories, (University of Sydney), Royal North Shore Hospital of Sydney, St. Leonards, N.S.W., Australia.
Ghosh
P
P
Taylor
T K
TK
Andrews
J L
JL
D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000375:Aging / Q000378:metabolism*; D002499:Centrifugation, Density Gradient; D002845:Chromatography; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007403:Intervertebral Disc / Q000737:chemistry* / Q000473:pathology; D007525:Isoelectric Focusing; D008159:Lumbar Vertebrae; D008875:Middle Aged; D008970:Molecular Weight; D015842:Serine Proteinase Inhibitors / Q000302:isolation & purification*
D015842:Serine Proteinase Inhibitors