Inhibitors of endogenous proteinases in the seeds of Scots pine, Pinus sylvestris

Extracts of resting pine seeds inhibited the proteinase activities present in extracts of endosperms of germinating seeds (hydrolysis of haemoglobin at pH 3.7 and hydrolysis of casein at pH 5.4 and 7.0). Heating the extracts of resting seeds at 60°C destroyed their own proteinase activity but their proteinase inhibitor activity decreased by only 25 to 30%. Some properties of the inhibitor(s) were studied using extracts treated at 60°C. The inhibitor activities were non-dialysable. the inhibition increased linearly with increasing inhibitor concentration up to 80% of total proteinase activity, and the maximal inhibition was 80% at pH 3.7. 90% at pH 5.4. and 97% at pH 7.0. The extracts of resting seeds did not inhibit the pepsin-like acid pine proteinase that accounts for a minor part of the proteolytic activity of endosperm extracts at pH 3.7. Neither did they have any effect on the acid pine carboxypeptidase or trypsin and chymotrypsin. Fresh extracts of endosperms of germinating seeds contained relatively high proteinase activity (assayed directly) and moderate inhibitor activity (assayed after treatment at 60°C). When fresh extracts were dialysed at 50°C for 48 h their proteinase activities increased considerably while the corresponding inhibitor activities disappeared. It is concluded that the decrease of inhibitors during dialysis is due to enzymatic inactivation and that the corresponding increase of proteinase activities is at least partly due to the destruction of the inhibitors.