Human Leukocyte Collagenase Research Articles

Activation of human leukocyte collagenase by compounds reacting with sulfhydryl groups

Human collagenase was partially purified from the granules of polymorphonuclear leukocytes by gel filtration, and the effects of two sulfhydryl reagents, N-ethylmaleimide and p-aminophenylmercuric acetate, on the enzyme activity were studied. The enzyme activity was assayed by incubating with soluble [ 14C]proline-labeled type I collagen, and the rate of collagen cleavage was quantitated by isolating the specific cleavage products by SDS-polyacrylamide gel electrophoresis. Results demonstrated that the collagenase, which was at first mostly in a latent form, was rapidly activated by these two sulfhydryl reagents. The enzyme activity, however, returned gradually to the control level in the presence of the sulfhydryl reagent or after removal of an excess of the reagent. The enzyme activity, after activation with N-ethylmaleimide, could be returned to the control level by the addition of a 2-fold molar excess of cysteine. The heat stability of the enzyme activity before and after activation by N-ethylmaleimide was also tested. The results indicated that the initial enzyme activity, before the activation, was stable at 60°C for at least 5 min, and the enzyme could be subsequently activated by N-ethylmaleimide to the same extent as an unheated control. If, however, the enzyme was first activated by incubating in the presence of N-ethylmaleimide and subsequently incubated at 60°C, a marked decrease in the enzyme activity as a result of the 5 min heating was noted. The results of the present study indicate that human leukocyte collagenase can be activated by compounds reacting with thiol groups.

Effects of human polymorphonuclear leukocyte collagenase on sub-component C1q of the first component of human complement

The similarities in the structure and properties of C1q and collagen prompted us to examine the susceptibility of C1q to human polymorphonuclear leukocyte collagenase. Incubation of C1q with a collagenase preparation resulted in no change in (1) the binding of C1q to immunoglobulin aggregates, (2) the hemolytic function of C1q as measured by reconstitution of C1q-depleted serum in immune hemolysis, or (3) the structural properties of C1q as revealed by gel electrophorettic patterns of the whole molecule or its polypeptide chains. In contrast, rapid inactivation and degradation of C1q was caused by leukocyte elastase. The collagenase preparation was, however, capable of cleaving reduced and carboxamidomethylated C1q into discrete fragments. This activity was attributed to a gelatinase present in the enzyme preparation since (1) the cleavage reaction was inhibited by denatured collagen but not by native collagen and (2) a collagenase fraction free of gelatinolytic activity could not degrade reduced and carboxamidomethylated C1q, while a gelatinase fraction devoid of collagenase activity retained the capacity to effect reduced and carboxamidomethylated C1q. Both collagenase and gelatinase activities were activated from the latent form by trypsin, and inhibited by EDTA. Therefore, it appears that native C1q lacks the structural features present in collagen which are recognized by leukocyte collagenase for hydrolytic action even though the denatured molecule still contains that region capable of being cleaved by gelatinase.

Human leukocyte collagenase: Characterization of enzyme kinetics by a new method

Human collagenase was partially purified from polymorphonuclear leukocytes, and a new method for assay of the collagenase activity was developed. The assay employs native radioactive collagen in soluble form as a substrate. The enzyme incubations are performed at 25°C which is below the melting temperatures of the cleavage products TCA and TCB, and these peptides are quantitatively recovered by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Employing this method, an apparent K m value of 1.04 × 10 −6 m for human leukocyte collagenase using type I collagen as a substrate was measured.

Inhibition of human leucocyte collagenase by some drugs used in the therapy of rheumatic diseases

Seven anti-inflammatory agents were tested for their possible inhibition of human leucocyte collagenase. Acetylsalicylic acid, indomethacin, phenylbutazone, sanocrisin and flufenamic acid were found to inhibit collagenase at concentrations which might be present in tissue during therapy. Hydrocortisone acetate and cyclophosphamide were ineffective.