Extraction of cartilage glycosaminoglycans: a comparison of ten solvents.

confirming that the binding of PI in the absence of Mn2+ is reversible. This was also demonstrated by the fact that it was possible to separate I 32PlPl completely from the enzyme by Sephadex G-100 gel filtration. When Pi followed by MnZ+ was added to the enzyme, dialysis did not restore activity. However, a substantial proportion of the activity reappeared during storage after dialysis, suggesting that this treatment had produced a conformational change in the enzyme from which it only slowly recovered, rather than that MnZ+ bound the PI irreversibly to the enzyme. This conclusion was supported by the fact that gel filtration removed [ 32P1Pi from the enzyme even when Mn2+ had been added. Inhibition of DNAase 4 by PI was not peculiar to the use of Mn2+ as the metal activator. Similar activity of this enzyme is achieved when Co2+, or Mg2+ plus Ca2+, is used in place of Mn2+, and higher activity with MnZ+ plus Ca2+. When these other ions, or ion combinations, were used, PI still inhibited strongly and with a similar dependence on addition sequence to that shown with Mn2+. Some other fungal nucleases have been reported to be inhibited by PI (see, e.g., Linn & Lehman, 1965; Holloman & Holliday, 1973). However, much higher concentrations of PI than those reported here were used. No effects of addition sequence seem to have been noted.