Human leukocyte collagenase: recent biochemical findings.
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Collagen↗
Biomedical subjects
K Suomalainen
Publications and source records attributed to K Suomalainen.
Gold sodium thiomalate activates latent human leukocyte collagenase.
Gold sodium thiomalate, a drug used widely in the therapy of rheumatoid arthritis, was found to be an activator of latent human polymorphonuclear leukocyte collagenase. The activation was demonstrated by two distinct and independent collagenase assays: by recording with a spectrophotometer at 227 nm the enzyme-induced increase in ultraviolet difference absorbance of native type I collagen connected to the cleavage of collagen at 37 degrees C [(1986) Eur. J. Biochem. 156, 1-4] and by SDS-polyacrylamide gel electrophoresis analysis of formation of specific products of collagen resulting from collagenase cleavage at 25 degrees C. Activation of latent collagenase by gold sodium thiomalate appeared to be of the same magnitude as by the known activator phenylmercuric chloride.
Electrophoresis, Polyacrylamide Gel↗
Hyperchromic effect of collagen induced by human collagenase.
Loss of the highly ordered triple-helix structure of native collagen on denaturation or enzymatic degradation involves a helix-to-coil transition, which can be seen as an increase at 227 nm in its ultraviolet difference absorption spectrum. We report here the successful use of this hyperchromic effect to quantify collagen in solution and to follow up the time-course of collagen degradation catalyzed by collagenase. Using 14C-labelled collagen substrate we show the excellent correlation between enzyme-induced increase in ultraviolet difference absorption and formation of specific cleavage products. The novel method was found to be suitable to characterize the enzymatic properties of human leukocyte collagenase. Activation of latent collagenase to the active enzyme could be followed continuously and an activation lag estimated.
Animals↗
Increased collagenase activity in human rheumatoid meniscus.
Collagenase activity of the knee joint menisci of patients suffering from rheumatoid arthritis was approximately 3-fold higher than that found in menisci of control patients. The mean collagenase activity in the macroscopically more diseased parts of the rheumatoid menisci was significantly higher than that in the less damaged areas. The specific degradation products resulting from the cleavage of human meniscoid type II collagen by rheumatoid meniscoid collagenase were demonstrated by SDS-polyacrylamide gel electrophoresis. Addition of N-ethylmaleimide, which activates latent mammalian collagenases, did not further increase collagenase activity in rheumatoid menisci. Thus in rheumatoid meniscus, collagenase may be synthesized and then activated, probably by proteolytic enzymes involved in the inflammatory reaction.
Arthritis, Rheumatoid↗
Partial purification and characterization of latent human leukocyte collagenase.
Latent and active collagenase were extracted from human polymorphonuclear leukocytes. Separation of the two forms of the enzyme was performed by gel filtration on Sepharose 6 B. The latent form of the enzyme was detected from chromatographic fractions after a brief treatment with trypsin or exposure of the fractions to the sulfhydryl reagent phenylmercuric chloride. Latent enzyme eluted before active enzyme from the column, indicating a higher apparent molecular weight. Partially purified latent enzyme exhibited an apparent molecular size of 70-75 kDa as estimated by gel filtration. A value of 50-55 kDa was obtained for active enzyme. Without activation the latent enzyme did not degrade soluble collagen substrate. This was demonstrated by a quantitative viscometric assay and also by sodium dodecyl sulfate polyacrylamide gel electrophoresis, when no typical cleavage products of collagen could be seen. Latent enzyme could not be obtained unless serine protease inhibitors were present during the extraction and purification procedures. The effects of the activators trypsin, phenylmercuric chloride, phenylmethyl sulfonyltrypsin, and N-ethylmaleimide on the latent human polymorphonuclear leukocyte collagenase were studied. Contrary to the suggestion that inactive proteases activate latent human polymorphonuclear leukocyte collagenase, the inactive phenylmethyl sulfonyl-trypsin could not activate latent collagenase.
Chromatography, Gel↗