Abstract
Abstract An enzyme discovered in the cell membrane fraction of Escherichia coli catalyzes the following reaction: CDP-diglyceride + H2O → CMP + phosphatidic acid The enzyme has been extracted from the membrane fraction with the nonionic detergent Triton X-100 and has been purified 115-fold. This novel hydrolase appears to have a high specificity, since it does not attack at a significant rate DPN, ATP, or CDP-choline, substrates that are rapidly split by nucleotide pyrophosphatase(s) of broad specificity previously described in plant and animal tissues. It also appears to be distinct from the periplasmic bacterial hydrolases specific for nucleoside diphosphate sugars. The CDP-diglyceride hydrolase is devoid of phospholipase D activity and is also unusual in that it is strongly inhibited by AMP.
Highlights
Preparation and Assay-Cells of E. coli B were grown on mineral medium 63 [3] with glycerol as carbon source, or were purchased as a frozen paste from Grain Processing Co., Muscatine, Iowa
The hydrolase described here has several novel properties when compared with the nucleotide pyrophosphatase(s) of plant and animal cells (g-12)
These are: (a) its localization in the membrane; (b) activation by detergent; (c) substrate specificity; and (d) inhibition by AMP. Glaser and his collaborators [14] have described a nucleotide pyrophosphatase from Bacillus subtilis 3610 with high specificity for CDP-glycerol. These workers, as well as Heppel and his co-workers, have studied the hydrolysis of nucleoside diphosphate sugars catalyzed by periplasmic enzymes of broad specificity, associated with 5’.nucleotidase activity in gram-positive [13] and in gram-negative organisms [15, 16]
Summary
An enzyme discovered in the cell membrane Escherichia coli catalyzes the following reaction: fraction of CDP-diglyceride + Hz0 -+ CMP + phosphatidic acid. The enzyme has been extracted from the membrane fraction with the nonionic detergent Triton X-100 and has been purified 115fold This novel hydrolase appears to have a high specificity, since it does not attack at a significant rate DPN, ATP, or CDP-choline, substrates that are rapidly split by nucleotide pyrophosphatase(s) of broad specificity previously described in plant and animal tissues. During the course of investigations on the enzymatic synthesis of phospholipids in cell-free extracts of E. coli, we have discovered a novel, membrane-bound enzyme of high specificity that catalyzes the hydrolysis of CDP-diglyceride at the pyrophosphate bond according to the following reaction: CDP-diglyceride + Hz0 -+ CMP + phosphatidic acid [1].
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