Enzymatic isolation of cells from bone: cytotoxic enzymes of bacterial collagenase.
The enzymatic isolation of cells from fetal rat calvaria is most effectively achieved with crude Clostridium histolyticum collagenase. However, this bacterial collagenase damages the cells during the digestion of the tissue. We have used cell density, as measured by isopycnic centrifugation on polysucrose gradients, as an indicator of cell damage. There are at least two enzymes in crude bacterial collagenase capable of damaging the cells in this tissue. One of these is clostripain that has been well characterized. The other cytotoxic enzyme is uncharacterized, and its effects are not evident until the clostripain activity has been inhibited by alpha-tosyl-lysyl chloromethane. The apparent activity of this second enzyme can be inhibited by withholding magnesium from the digestion medium and by increasing the potassium concentration of the digestion medium.
D000818:Animals; D001842:Bone and Bones / Q000166:cytology*; D002469:Cell Separation / Q000379:methods*; D003546:Cysteine Endopeptidases; D003012:Microbial Collagenase / Q000302:isolation & purification / Q000494:pharmacology; D011480:Protease Inhibitors; D051381:Rats; D014107:Tosyllysine Chloromethyl Ketone; D014357:Trypsin
D011480:Protease Inhibitors; D014107:Tosyllysine Chloromethyl Ketone; D014357:Trypsin; D003546:Cysteine Endopeptidases; C019913:clostripain; D003012:Microbial Collagenase