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A Simple Method for Estimating Intactness of Spinach Leaf Protoplasts by Glycolate Oxidase Assay ¹

Research Institute for Biochemical Regulation, School of Agriculture, Nagoya University, Chikusa, Nagoya 464, Japan, Physiologie Cellulaire Végétale, CEN-6 and USM-685 38041, Grenoble Cedex, France

A method was developed for the quantitative analysis of intactness of spinach leaf protoplasts using glycolate oxidase activity as an index. Since glycolate does not penetrate into protoplasts at neutral pH, the increase of O₂ consumption by the addition of glycolate to protoplast suspension was due to the glycolate oxidase activity released from damaged protoplasts. The proportion of damaged protoplasts in the whole preparation was calculated from the ratio of released and total glycolate oxidase activity. Freshly prepared spinach leaf protoplasts were found to be 80 to 90% intact as estimated by the method. The effect of osmolarity on the respiratory activities of spinach leaf protoplasts was also examined by applying the same principle.

¹ Supported in part by grants from the Japanese Ministry of Education, Science and Culture (Mombusho) to M.N. and T.A. and from the Nissan Science Foundation (Tokyo) to M.N. This is paper No. 66 in the series `Structure and Function of Chloroplast Proteins.'