Purification of CMP-N-acetylneuraminic acid synthetase from bovine anterior pituitary glands.
CMP-beta-N-acetylneuraminic acid (CMP-neuNAc) is the substrate for the sialylation of glycoconjugates by sialyltransferases in microbes and higher eukaryotes. CMP-neuNAc synthetase catalyzes the formation of this substrate, CMP-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 of the 52 kDa protein band was confirmed by native gel electrophoresis in that the position of the enzyme activity in gel slices coincided with the position of major bands in the stained gel. Photoaffinity labeling with 125I-ASA-CDP ethanolamine resulted in the modification of a 52 kDa polypeptide that was partially protected against modification by the substrate CTP. Enzyme activity in crude fractions could be adsorbed onto an immunoadsorbent prepared from antibody against the purified 52 kDa protein. Taken together these data suggest that the 52 kDa polypeptide purified by this procedure described in this report is indeed CMP-neuNAc synthetase. The active enzyme chromatographed on a gel filtration column at 158 kDa suggesting it exists in its native form as an oligomer.
Vionnet
J
J
Laboratory of Bacterial Toxins, Division of Bacterial Products, OVRR, CBER, FDA, 8800 Rockville Pike, Bethesda, MD 20892, USA.
Concepcion
N
N
Warner
T
T
Zapata
G
G
Hanover
J
J
Vann
W F
WF
D000818:Animals; D002417:Cattle; D002467:Cell Nucleus / Q000201:enzymology; D002846:Chromatography, Affinity; D004247:DNA / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D007526:Isoelectric Point; D008970:Molecular Weight; D000216:N-Acylneuraminate Cytidylyltransferase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D010903:Pituitary Gland, Anterior / Q000201:enzymology*
D004247:DNA; D000216:N-Acylneuraminate Cytidylyltransferase