Purification of CMP-N-acetylneuraminic acid synthetase from bovine anterior pituitary glands

CMP-beta-N-acetylneuraminic acid (CMP-neuNAc) is the substrate for the sial ylation of glycoconjugates by sialyltransferases in microbes and higher euk aryotes. CMP-neuNAc synthetase catalyzes the formation of this substrate, C MP-neuNAc, from CTP and neuNAc. In this report we describe the purification of CMP-neuNAc synthetase from bovine anterior pituitary glands. The enzyme was purified by ion exchange, gel filtration, and affinity chromatography, The protein was homogeneous on SDS-PAGE with a molecular weight of 52 kDa, a subunit size similar to that of the E.coli K1 (48.6 kDa), The identity o f the 52 kDa protein band was confirmed by native gel electrophoresis in th at the position of the enzyme activity in gel slices coincided with the pos ition of major bands in the stained gel. Photoaffinity labeling with I-125- ASA-CDP ethanolamine resulted in the modification of a 52 kDa polypeptide t hat was partially protected against modification by the substrate CTP, Enzy me activity in crude fractions could be adsorbed onto an immunoadsorbent pr epared from antibody against the purified 52 kDa protein. Taken together th ese data suggest that the 52 kDa polypeptide purified by this procedure des cribed in this report is indeed CMP-neuNAc synthetase. The active enzyme ch romatographed on a gel filtration column at 158 kDa suggesting it exists in its native form as an oligomer.