Partial purification of collagenase and gelatinase from human polymorphonuclear leucocytes. Analysis of their actions on soluble and insoluble collagens

Abstract

The separation and further purification of human polymorphonuclear-leucocyte collagenase and gelatinase, using modifications of the method of Cawston & Tyler [(1979) Biochem J. 183, 647-656], are described. The final preparations yielded collagenase of specific activity 260 units/mg and gelatinase of specific activity 13 000 units/mg. Gelatinase was purified to apparent homogeneity in a latent form, and analysis of the activation of 125I-labelled latent enzyme by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and gel-filtration techniques suggested that no peptide material was lost on conversion into the active form. The purified natural inhibitors alpha 2-macroglobulin, tissue inhibitor of metalloproteinases ('TIMP') and amniotic-fluid inhibitor of metalloproteinases all inhibited the two polymorphonuclear-leucocyte metalloproteinases, but the last two inhibitors were slow to act and complete inhibition was difficult to attain. Collagenase degraded soluble types I and III collagen equally efficiently, but soluble type II collagen less well. Gelatinase alone had little activity on these substrates, although it enhanced the action of collagenase. Gelatinase was capable of degrading soluble types IV and V collagen at 25 degrees C, whereas collagenase was only active at higher temperatures when the collagens were susceptible to trypsin activity. By using tissue preparations of insoluble collagens (type I, II or IV) the activity of leucocyte collagenase was low and gelatinase activity was negligible, as measured by the solubilization of hydroxyproline-containing material. The two enzymes together were two or three times more effective in the degradation of these insoluble collagens.

Biomechanical regulation of matrix metalloproteinase‐9 in cultured chondrocytes

“…MMP-9 expression is increased in degenerated cartilage (51), especially in the mid-deep layers (24). Because MMP-9 can degrade various cartilage matrix components, including denatured collagen, type-XI collagen, aggrecan, and link protein (22,34,54,55), it likely contributes to the progressive erosion of cartilage in osteoarthritis. The relative cxtent of specific degradation pathways-for example, resulting from deleterious loading-leading to extensive, and potentially irreversible, cartilage damage in osteoarthritis remains to be established, as does the role of mechanical stimuli in eliciting catabolic responses.…”

“…MMP-9 can degrade denatured collagen, type-XI collagen, aggrecan, link protein. and other matrix molecules (22,34,54,55). Normal human and rabbit articular cartilage and cultured chondrocytes express little or undctectable levels of MMP-9 under basal conditions, but the gene is induced by treatment with inflammatory cytokines such as interleukin-la (IL-la) (32,35,43,51,57,64).…”

Identification of matrix metalloproteinases 3 (stromelysin-1) and 9 (gelatinase B) in abdominal aortic aneurysm.

“…34 MMP-9 also facilitates the action of MMP-1 (collagenase) on native type I and type III collagens, which are major constituents of the vascular wall. 35 In addition, Okada et al 25 report that MMP-9 has some ability to degrade fibrillar collagens. The presence of MMP-1 in aneurysmal aorta has not been firmly established.…”

Metalloproteinase inhibitors from bovine cartilage and body fluids

“…Crude rabbit skin collagenase, gelatinase and proteoglycanase were from concentrated rabbit skin medium and bovine collagenase and gelatinase were from bovine cartilage disc medium. Human leucocyte collagenase and gelatinase were prepared as published [16]. Purified rabbit bone gelatinase was a gift from C. Poll, Strangeways Laboratory (unpublished) and purified protcoglycanase was prepared by our published method [17].…”