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Freezing Injury and Phospholipid Degradation in Vivo in Woody Plant Cells

III. Effects of Freezing on Activity of Membrane-bound Phospholipase D in Microsome-enriched Membranes ¹

^a The Institute of Low Temperature Science, Hokkaido University, Sapporo, Japan 060

Freeze-thawing of microsome-enriched membranes from living bark tissues of black locust trees, especially those from less hardy tissues, caused a drastic increase in sensitivity to Ca²⁺ and a complete loss of the regulatory action of Mg²⁺ in membrane-bound phospholipase D activity with endogenous (membrane-bound) substrates. Also, the freeze-thaw cycle made phospholipase D in these membranes more resistant to digestion by proteases. Thus, the regulatory properties of the membrane-bound phospholipase D seem to be dependent on the nature of the membranes and on the interaction between the enzyme and membranes as well. The alteration of regulatory properties by freezing was protected by sucrose, at lower concentrations, and more effectively for membranes from hardy tissues than for membranes from less hardy tissue. Addition of partially purified soluble phospholipase D to the reaction system containing membranes caused only a slight stimulation of the degradation of endogenous phospholipids. Phospholipid degradation in vivo during freezing of less hardy tissue may be catalyzed mainly by the bound enzyme. Disintegration of the tonoplast, however, besides releasing soluble phospholipase D into the cytosol, would release organic acids (lowering the pH) and free Ca²⁺. Both factors would stimulate drastically the membrane-bound phospholipase D, causing degradation of membrane phospholipids.