Neutral proteinases of the human intervertebral disc

Abstract

Disc tissue consisting of pooled annuli fibrosus and nuclei pulposus from the cadaver of an adolescent aged 19 years was extracted with 4.0 M Gu-HCl. Proteins of low buoyant density ( ϱ ≤ 1.38 g/ml) containing the disc enzymes and inhibitors were separated from proteoglycans of high buoyant density ( ϱ ≥ 1.50 g/ml) by density gradient ultracentrifugation. Sephadex G-75F gel chromatography followed by trypsin affinity chromatography was then used to resolve disc proteolytic and trypsin inhibitory activities. The results obtained were strongly suggestive of the presence of a high molecular weight zymogen which upon activation generated a population of smaller molecular weight proteinases. The disc proteinases obtained by this process showed similar properties in terms of: (a) their pH optima (7.4–7.6); (b) their inhibition patterns by class-specific proteinase inhibitors; (c) their variation of activity as a function of NaCl and lysine concentrations; and (d) the hydrodynamic size of their proteoglycan degradation products. The activated disc neutral proteinase demonstrated many characteristics in common with plasmin; however, unlike the latter, the disc proteinases also showed some calcium dependence.