Harold M. Fullmer

Collagenase: Localization in Polymorphonuclear Leukocyte Granules in the Rabbit

Polymorphonuclear leukocyte granules were submitted to zonal fractionation through a discontinuous sucrose gradient. Azurophilic and specific granules were enzymatically characterized by peroxidase and alkaline phosphatase activity, respectively. The enzymes formed modal distributions like those reported by others. Collagenase activity was consistently associated with the specific granules containing alkaline phosphatase.

Activation of fibroblast procollagenase by mast cell proteases

Proteases capable of activating procollagenase from gingiva and from fibroblast and macrophage monolayer cultures were harvested from homogenates of canine tumor mast cells. The mast cell proteases lysed casein and Azocoll but not native collagen. In low salt concentrations the enzymes existed at high molecular weight complexes, which were dissociated by increasing the salt concentration above 1.0 M (NaCl, KCl). Gel filtration in 1.4 M KCl separated the protease activity into three peaks, all of which activated procollagenase. Two of the enzymes showed substrate specificities (hydrolysis of p-tosyl-L-arginine methyl ester and benzoyl-tyrosine ethyl ester) and reactive center reactivities similar to pancreatic trypsin and chymotrypsin. Based on gel filtration, apparent molecular weights of 160 000 (p-tosyl-L-arginine methyl ester esterase), 90 000 (main procollagenase activator) and 36 000 benzoyl-tyrosine ethyl ester esterase) were determined. Activation of procollagenase resulted in a 18-20 000 decrease of the molecular weight. The activation was directly related to the amount of activator added within certain limits. Further addition of activator resulted in proteolytic inactivation of collagenase.

A reproducible quantitative collagenase radiofibril assay

Abstract A radioactive fibril assay for collagenase utilizing [ 14 C]glycine-labeled chick calvaria collagen is presented. Several incubation modifications are evaluated to obtain increased sensitivity and low statistical error. The modifications described may also be applicable to other fibril assay systems.

Fibroblastic origin of bovine gingival collagenase

Abstract The site of collagenase production in explants of bovine gingiva was located by tissue culture techniques. Collagenase was released from the juxta-epithelial connective tissue, but not from the epithelium. Addition of serum to the explants resulted in outgrowth of a single cell type, which was identified as a fibroblast on the basis of culture morphology and light and electron-microscopic characteristics. The fibroblasts elaborated a collagenase that was indistinguishable from the enzyme harvested from whole bovine gingival explants. The enzymes, both of which were released in latent form, had the same molecular size and had common antigenic sites.

Procollagenase from bovine gingiva.

1. Collagenase (EC 3.4.24.3) is released from bovine gingival explants in vitro as a zymogen. The zymogen does not hydrolyze collagen and does not form a complex with alpha2-macroglobulin (alpha2-M). It elutes in gel filtration with an apparent molecular weight of approx. 80 000. 2. Incubation of the zymogen with trypsin results in a 15 000-20 000 dalton decrease in molecular weight and imparts to the enzyme the ability to hydrolyze collagen and to form a complex with alppha2-M. 3. The zymogen can be completely separated from the active enzyme to alpha2-M. Likewise, the zymogen can be harvested from cultures supplemented with serum.

In vivo and in vitro stimulation of collagenase production by rabbit alveolar macrophages.

Abstract Rabbit alveolar macrophages, harvested by lavage, were cultured for 4 days in Dulbeccos modified Eagle medium and the culture fluids assayed for collagenase activity. Stimulation of the macrophages in vivo by injection of Freunds complete adjuvant or vitro by addition of a soluble bovine lymphocyte extract led to a 5–10 fold increase in the production of collagenase. The major part of the collagenase present in the culture fluids was latent and could be activated by brief exposure to trypsin.

Activation of latent bovine gingival collagenase

Abstract Latent collagenase from primary cultures of bovine gingiva was activated by brief exposure to trypsin and to a lesser extent by thiocyanate. The data suggest that activation by trypsin involves two distinct mechanisms: release of collagenase activity from high molecular weight inhibitor complexes and activation of latent collagenase (possibly a procollagenase) of approximately the same molecular size as active collagenase. Inhibition experiments confirmed that collagenase activity can be recovered from inactive inhibitor complexes, formed by the addition of whole serum or α 2 -macroglobulin alone, after dialysis against thiocyanate or by brief exposure to trypsin.

Rabbit alveolar macrophage collagenase: evidence of polymeric forms.

Collagenase harvested in vitro from rabbit alveolar macrophages eluted in gel chromatography corresponding to apparent molecular weights of 45 000, 85 000, and 165 000. Reversible changes from one molecular weight to another in low salt concentration and predominance of the 45 000 species in salt concentrations above 1.0 M (NaCl, KCl) suggest that the higher molecular weights represent polymeric forms of collagenase.