Extraction of collagenase from the involuting rat uterus.
Collagenase (EC 3.4.24.3) activity can be measured directly in homogenates of the involuting rat uterus. Latent forms of collagenase are activated by a brief exposure to trypsin; trypsin activity is then blocked with soybean trypsin inhibitor. Homogenizing conditions have been developed that permit 90-95% recovery of the total active and latent collagenase activity in a 6000 X g pellet, where it is presumably bound to its collagen substrate. This insoluble activity can then be extracted by heating to 60 degrees C for 4 min in 0.04 M Tris - HCl buffer, pH 7.5, containing 0.1 M CaCl2. Methods are presented for the estimation of the recovery of collagenase in the extracts; this approximates 65-70% of the total. Small amounts of activity can also be extracted from rat liver and kidney. This extraction procedure should be of use in purifying collagenase without culturing the enzyme-producing tissue and in the direct assay of tissue collagenase activity. The activity extracted from rat uterus has been proven to be collagenase by its characteristic pattern of collagen breakdown products on disc electrophoresis and by the split of tropocollagen at interband 41 as shown by electron microscopy of reconstituted fragments. The activity is inhibited by EDTA, and this inhibition is not reversed by calcium or zinc ions.
D000818:Animals; D002118:Calcium; D004492:Edetic Acid / Q000494:pharmacology; D005260:Female; D006358:Hot Temperature; D008722:Methods; D003012:Microbial Collagenase / Q000302:isolation & purification* / Q000378:metabolism; D011247:Pregnancy; D051381:Rats; D014357:Trypsin; D014599:Uterus / Q000201:enzymology*; D015032:Zinc / Q000494:pharmacology
D004492:Edetic Acid; D014357:Trypsin; D003012:Microbial Collagenase; D015032:Zinc; D002118:Calcium