Synthesis of biologically active spin-labelled radioactive cytidine diphosphodiglyceride, a novel probe for biological membranes

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Abstract
A versatile synthesis of spin-labeled radioactive cytidine diphospho-sn-1,2-diacylglycerol (CDP-diglyceride) was developed based on the combination of the enzymatic acylation of radioactive sn-glycero-3-phosphate with 12-doxyl stearic acid and the chemical conversion of the obtained spin-labeled radioactive phosphatidic acid with cytidine monophosphomorpholidate into spin-labeled radioactive CDP-diglyceride. The method for the isolation and purification of the latter compound was described. This obtained CDP-[2-3H]diglyceride contained 10% of fatty acids of paramagnetic nature, presumably present as a covalently bound 12-doxyl stearic acid esters. The biological activity was tested by using the synthesized compound as a substrate in the mitochondrial biosynthesis of phosphatidylglycerol. Spin-labeled CDP-[2-3H]diglyceride prepared as described can be converted in the presence of sn-[2-14C]-glycero-3-phosphate into a spin-labeled [2-3H, 2''-14C]phosphatidylglycerol with isolated rat liver mitochondria, establishing that the site of its utilization is identical with the site of phosphatidylglycerol synthesis in isolated mitochondria, i.e., inner mitochondrial membrane. The synthesized spin-labeled CDP-diglyceride can be used as a specific probe for the spin- and radioactive covalent labeling of polyglycerophosphatides of mitochondrial membranes. Some implications and further possibilities in the study of biological membranes using the spin-labeled radioactive CDP-diglyceride are discussed.