Mouse Bone Collagenase

Abstract

Animal collagenase, first isolated from tadpoles (1, 2), has also been detected in a number of other tissues (3-14). To date, however, only the enzymes obtained from tadpole fin (15, 16), rat uterus (14), rheumatoid synovial tissue (12), and human skin (13) have been partially purified and characterized, and their mode of action in degrading the collagen macromolecule studied.

If tissue collagenase is physiologically important and plays a significant role in tissue collagen degradation, one would expect considerable quantities of such an enzyme to be synthesized in bone, since the turnover of collagen in bone is amongst the highest of any of the collagenous tissues (17). Indeed, collagenase activity has been observed in tissue culture media of bone cultured in vitro from animals which had received parathyroid hormone (3) and which had been undergoing resorption in vim, and in the tissue culture media of bone actively undergoing bone resorption in vitro (4). In the latter study, increased collagenolytic activity was shown to parallel the increased rate of bone resorption when parathyroid hormone, and parathyroid hormone and heparin were added to the tissue culture medium (4). The present communication deals with the isolation, partial purification, and mode of action of mouse bone collagenase, obtained from the tissue culture media of living bone cultured in vitro in the presence of heparin (18).

Methods. Tissue culture of bone. Tibiae of 5-day old Swiss albino mice of the Webster strain were removed aseptically and muscle and connective tissue carefully removed. Immediately thereafter, four bones were placed in a roller tube containing 2 ml of tissue culture medium to which was added 0.1 ml of heparin (100 units) dissolved in Gey's solution, and the tubes were gassed with 95% O2 and 5% COa for approximately 1 min. The tissue culture medium was composed of 95 ml of mammalian Tyrode solution, 1 ml each of 100 times concentrated amino acid mixture, 100 times concentrated vitamin mixture, and 2'00 mM L-glutamine (all from Microbiological Associates, Inc., Bethesda, Maryland), and 2 ml containing 5000 units each of penicillin and streptomycin.