The enzymatic synthesis of inositol monophosphatide.

Abstract

The enzymatic synthesis of inositol monophosphatide has been described in reports by Agranoff et al. (l), Thompson et al. (2), and Paulus and Kennedy (3, 4). An enzymatic reaction in which free inositol is incorporated into inositol phosphatide, stimulated by the addition of any of a number of cytidine nucleotides, including cytidine 5’-monophosphate and cytidine diphosphate choline, was fnst observed by Agranoff et al. (1) and confirmed by other workers (24). To explain the effect of CDP-choline, a mechanism was postulated (1) for the biosynthesis of inositol monophosphatide involving a reaction between CDP-choline and phosphatidic acid, with the formation of cytidine diphosphate diglyceride (Fig. 1). Evidence for the formation of a chloroform-soluble compound from radioactive CMP was obtained in an experiment by Agranoff et al. (1). The amounts of product obtained were too small to permit satisfactory characterization, and it was not shown that this transformation was in fact related to the incorporation of inositol into lipid. Nevertheless, this experiment provided an important clue to the nature of the cytidine nucleotide intermediate. In experiments to be described in this paper, we have found that free inositol may be incorporated into lipid as a result of an enzymatic exchange reaction with inositol phosphatide present in the enzyme preparation. In the presence of saturating amounts of manganese ions, this exchange takes place at maximal rates in the absence of any added cytidine nucleotides. In the presence of low amounts of manganese ions, a stimulation of the exchange by cytidine nucleotides may be observed, similar to the effects reported by Agranoff et al. (1) and Thompson et al. (2). It appears that this exchange is not directly related to the synthesis de novo of inositol monophosphatide. In the synthesis de novo of inositol monophosphatide, on the other hand, n-a-glycerophosphate-P32 is converted to inositol monophosphatide by a pathway in which the phosphatide-phosphorus is derived from L-oc-glycerophosphate, and is thus strikingly different from the enzymatic synthesis of lecithin and of phosphatidyl ethanolamine (5). The synthesis de nova of inositol monophosphatide from n-cr-glycerophosphate specifically requires CTP. CDP-choline is inactive; this and other evidence rules out the postulated role (1) of CDP-choline in the biosynthesis of inositol monophosphatide in our system. Evidence will be presented in support of the following reaction scheme accounting for the synthesis of inositol monophosphatide in particulate enzymes derived from liver:

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