The subcellular distribution of soluble protease in anthesis-stage, anthocyanin-containing Hippeastrum cv. Dutch Red Hybrid petal protoplasts has been reevaluated and that of Triticum aestivum L. var. Red Coat leaf protoplasts determined using 125I-fibrin as a protease substrate and improved methods for protoplast and vacuole volume estimation. Results indicate that about 20% of the Hippeastrum petal-soluble protease and about 90% of the wheat leaf-soluble protease can be assigned to the vacuole. Protoplast isolation enzyme labeled with 125I has been used to assess the efficiency of removing isolation enzyme from protoplasts by repeated washing and by separation of protoplasts from debris using density centrifugation. Results of these studies suggest that protoplasts prepared by both methods retain low levels of isolation enzyme. However, when protoplasts prepared by either method were lysed with washing medium lacking osmoticum, little isolation enzyme contaminated the lysates.
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