leupeptins and 4-aminophenylmercuriacetate

Novel gelatinolytic activities in both latent and active forms were detected in the normal organs of rat by gelatin zymography. Multiple active bands were detected in the extracts from the skin, jejunum, muscle, and kidney without any activation. These activities were inhibited by 1,10-phenanthroline or leupeptin, nor by E64, suggesting that these activities were derived from metallo-proteinases or serine-proteinases. Some gelatinolytic active bands were newly induced or enhanced by p-aminophenylmercuric acetate. These results suggest that matrix degrading activities due to metallo- and serine-proteinases were constitutively expressed in various rat normal organs.

Topics: Animals; Cattle; Enzyme Inhibitors; Gelatin; Leupeptins; Male; Metalloendopeptidases; Phenanthrolines; Phenylmercuric Acetate; Rats; Rats, Wistar; Serine Endopeptidases

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 (p less than or equal to 1.38 g/ml) containing the disc enzymes and inhibitors were separated from proteoglycans of high buoyant density (p greater than or equal to 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: their pH optima (7.4-7.6); their inhibition patterns by class-specific proteinase inhibitors; their variation of activity as a function of NaCl and lysine concentrations; and 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.

Topics: Adolescent; Adult; Chromatography; Chromatography, Gel; Endopeptidases; Enzyme Activation; Female; Humans; Intervertebral Disc; Leupeptins; Male; Phenylmercuric Acetate; Protease Inhibitors; Sodium Chloride; Ultracentrifugation