Yoshida S
The Institute of Low Temperature Science, Hokkaido University, Sapporo, Japan 060.
Plant Physiol. 1979 Aug;64(2):252-6. doi: 10.1104/pp.64.2.252.
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 Ca(2+) and a complete loss of the regulatory action of Mg(2+) 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 Ca(2+). Both factors would stimulate drastically the membrane-bound phospholipase D, causing degradation of membrane phospholipids.
对刺槐活树皮组织中富含微粒体的膜进行冻融处理，尤其是对那些来自抗寒能力较弱组织的膜，会导致其对Ca(2+)的敏感性急剧增加，并且在以内源性（膜结合）底物进行膜结合磷脂酶D活性反应时，Mg(2+)的调节作用完全丧失。此外，冻融循环使这些膜中的磷脂酶D对蛋白酶的消化更具抗性。因此，膜结合磷脂酶D的调节特性似乎取决于膜的性质以及酶与膜之间的相互作用。较低浓度的蔗糖可保护冻融导致的调节特性改变，且对来自抗寒组织的膜的保护效果比对来自抗寒能力较弱组织的膜更有效。向含有膜的反应体系中添加部分纯化的可溶性磷脂酶D只会轻微刺激内源性磷脂的降解。抗寒能力较弱组织在冷冻过程中体内的磷脂降解可能主要由结合型酶催化。然而，液泡膜的解体除了会将可溶性磷脂酶D释放到细胞质溶胶中外，还会释放有机酸（降低pH值）和游离Ca(2+)。这两个因素都会极大地刺激膜结合磷脂酶D，导致膜磷脂降解。
