Purification of Rheumatoid Synovial Collagenase and Its Action on Soluble and Insoluble Collagen

Abstract

1. The neutral collagenase released into the culture medium by explants of rheumatoid synovial tissue has been purified by ultrafiltration and column chromatography, utilising Sephadex G-200, Sephadex QAE A-50 and Sephadex G-100 superfine.2. The final collagenase preparation had a specific activity against thermally reconstituted collagen fibrils of 312 l g collagen degraded min-' mg enzyme protein-', representing more than a 1000-fold increase over that of the active culture medium.3. Electrophoresis in polyacrylamide disc-gels with and without sodium dodecyl sulphate showed the enzyme to migrate as a single protein band. Elution experiments from polyacrylamide gels and chromatography columns have provided no evidence for the existence of more than one collagenase.4. The molecular weight of the enzyme, as determined by dodecylsulphate-polyacrylamide gel electrophoresis, was 33 000.5. Data obtained from studies with the ion-exchange resin and from gel electrophoresis in acid and alkaline buffer systems suggested a basically charged enzyme.6. It did not hydrolyse the synthetic collagen peptide Pz-Pro-Leu-Gly-Pro-D-Arg and non-specific protease activity was absent.7. The collagenase attacked undenatured collagen in solution at 25 "C resulting in a 58 % loss of viscosity and producing the two characteristic products TCA (3/4) and TCB (1/4).8. At 37 "C and pH 8.0 both reconstituted collagen fibrils and gelatin were degraded to peptides of less than 10000 molecular weight.9. As judged by the release of soluble hydroxyproline peptides and electron microscopic appearances the enzyme degraded human insoluble collagens derived from tendon and soft juxta-articular tissues although rates of attack were less than with reconstituted fibrils.10. The data suggests that pure rheumatoid synovial collagenase at 37 "C and neutral pH can degrade gelatin, reconstituted fibrils and insoluble collagens without the intervention of non-specific proteases.11. The different susceptibilities of various collagenous substrates to collagenase attack are discussed.Knowledge of the action of purified human collagenase is central to our understanding of its role in vivo and to any considerations that its specific inhibition might offer a therapeutic approach to the management of diseases characterised by disordered collagen metabolism. Since the first purification of an animal collagenase from tadpole tailfin cultures by Nagai et al. in 1966 [l] a number of approaches to the purification of animal collagenases have been adopted which include gel filtration [2 -41, ion-exchange chromatography [5,6] and affinity chromatography [7-101. In the present study rheumatoid synovial collagenase from tissue culture medium has been purified by a procedure involving a series of chromatographic steps designed to facilitate a quick and effective separation of the enzyme. Since previous attempts to study the properties of the synovial enzyme have largely utilised impure preparations, or have been incomplete Eur.

Collagenase and collagenase inhibitors in osteoarthritic and normal cartilage.

“…The pattern of breakdown of collagen at 370C to dialyzable fragments which we observed in electrophoresis and measured in the dialysate has recently been reported for rheumatoid synovial collagenase (24). It was noted that this reaction could proceed to complete digestion of the collagen where break-2 A a FIGURE 4 Electrophoresis of collagen (A) and collagen incubated with collagenase (B) from osteoarthritic cartilage at 250C.…”

Chemical and Enzymatic Characterization of the Collagenase from the Insect Hypodearma lineatum

“…Moreover, the values reported with identical units for collagenases of different species vary widely. Specific activity of 32 and 312 pg collagen degraded min-' (mg enzyme)-' respectively were obtained for collagenases from human skin [42] and human rheumatoid synovium [43] with reconstituted guinea pig skin collagen as substrate. Much higher values of 3583 and 4020 pg min-' mg-I were found for the collagenases from rabbit tumour [44] and fiddler crab hepatopancreas [7].…”

Activation of matrix metalloproteinase 3 (stromelysin) and matrix metalloproteinase 2 (‘gelatinase’) by human neutrophil elastase and cathepsin G

“…Rheumatoid synovial fibroblasts in culture secrete three distinct matrix metalloproteinases (MMPs): MMP-1 corresponds to collagenase (EC 3.4.24.7) [1], MMP-2 to 'gelatinase' and type IV collagenase [2][3][4][5] and MMP-3 to stromelysin [6][7][8]. Collagenase digests type I, II, III and X [9] collagens.…”