Determination of proteases in isolated washed protoplasts: Inactivation of proteases in cell wall-degrading enzyme mixtures used in protoplast isolation

Abstract

Protease activity was determined in oat leaf protoplasts after isolation and washing. Protoplasts were isolated with a cellulase R-10, macerozyme R-10 mixture, which contains high proteolytic activity. Proteases in this cell wall-degrading enzyme mixture were inactivated by heating the enzyme preparation at 50°C for 10 min at pH 6.5. This treatment did not impair the cell wall-degrading activity. Protease activity in protoplasts isolated with heated enzyme was similar after washing to that in protoplasts isolated with untreated enzymes. This provided proof that contaminating proteases were effectively removed during protoplast washing and that the protease activity measured in isolated protoplasts was derived from the protoplasts themselves.