Subcellular localization of sugar nucleotide synthetases.

Sugar nucleotides are Drecursors of sugar moieties in obtained bv others in brain and kidney (Gielen et al., 1970 glycGlipids and glycoproteins and thus may have a regulatory role in the metabolism of these macromolecules.We have therefore studied the subcellular localization of four sugar nucleotide synthetases.The pyrophosphorylases of GDP-fucose, GDP-mannose, and UDP-glucose were detected only in the cytoplasm; in contrast, at least 85% of the CMP-N-acetylneuraminic acid (CMP-NeuAc) synthetase was found in the nucleus."he latter conclusion was reached by assaying CMP-NeuAC synthetase activity i n nuclei and cytoplasm of cells fractionated by a nonaqueous procedure and in karyoplasts and cytoplasts from cells enucleated with Cytochalasin B. In addition, cytoplasts were able to synthesize in situ only 20% as much CMP-NeuAc from free NeuAc as nucleated cells; the same enucleated cells, however, synthesized GDP-fucose from free fucose at levels comparable to nucleated cells.The nuclear CMP-NeuAc synthetase activity could not be solubilized by treatment of the nuclei with a mixture of 2% Triton X-100 and 1% sodium deoxycholate, conditions which solubilize both the outer and inner nuclear membranes and the nuclear pores.These results strongly suggest that the activities of CMP-NeuAc synthetase and GDP-fucose pyrophosphorylase which are assayed in vitro are those catalyzing the reactions in vivo.Sugar nucleotides are intermediates in the biosynthesis of glycoproteins and glycolipids and may therefore play a regulatory role in their metabolism.Current knowledge of the subcellular localization of enzymes that catalyze the synthesis of sugar nucleotides is rather limited, and only recently have studies begun on how sugar nucleotides reach the sites where the sugar moieties are transferred to macromolecules (Carey et al., 1980).We have become interested in the subcellular localization of sugar nucleotide synthetases in general and CMP-NeuAcl synthetase in particular as a result of our recent characterization of this sugar nucleotide from mammalian tissues (Carey and Hirschberg, 1979) and the previous studies of Kean (1970).Kean showed that between 18% and 50% of the CMP-NeuAc synthetase activity of liver, retina, and kidney was associated with nuclei.Similar results were also