Abstract
A UDP-Gal:N-acetylglucosamine beta(1,4)-galactosyltransferase which catalyzes the synthesis of beta-D-Gal(1,4)-D-GlcNAc units has been purified 17,560-fold from Ehrlich tumor cells to apparent electrophoretic homogeneity. The enzyme appears to be a monomeric protein with Mr = 56,000-58,000. Enzymatic activity requires the presence of MnCl2, is stimulated by detergent, and exhibits a pH optimum at 6.9. The Km values for GlcNAc and UDP-Gal are 1.89 and 0.046 mM, respectively. The Ehrlich cell beta-galactosyltransferase acts efficiently on glycoproteins and glycolipids terminating in GlcNAc, but is inactive toward glycoconjugates possessing terminal GalNAc units. The oligosaccharides beta-D-GlcNAc(1,3)-D-Gal and beta-D-GlcNAc(1,3)[beta-D-GlcNAc(1,6)]-D-Gal are good acceptors for the beta-galactosyltransferase from Ehrlich cells, suggesting that the enzyme may participate in the biosynthesis of i/I structures. In addition, other linear and branched sugars presenting GlcNAc residues at their nonreducing termini also act as acceptors for the enzyme. The activity of Ehrlich cell beta-galactosyltransferase both in the presence and absence of alpha-lactalbumin has been studied using a series of derivatives of Glc and GlcNAc which were substituted at various positions of the pyranose ring. This study has provided a map of the molecular contacts necessary for enzymatic activity in the presence and in the absence of alpha-lactalbumin.
Highlights
A UDP-Ga1:N-acetylglucosamineB( l,l)-galactosyl- protein is a membrane-bound enzyme localized in the trans transferasewhich catalyzes the synthesis of 8-D- cisternae of the Golgi apparatus (3)
Efforts to clone requires the presence MofnC12,is stimulated by deter- the 8-galactosyltransferase have resulted in the isolation of gent, and exhibits a pH optimum at 6.9.The K, values cDNAs encoding the carboxyl-terminal portion of the bovine forGlcNAcandUDP-Gal are 1.89 and 0.046 mM, enzyme (13, 14), and afull-length cDNA clone of the human respectively
19) have revealed the presence of a high affinity metal site for Mn2+(Kdin the micromolar range) which is involved in GlcNAc(l,3)[~-D-GlcNAc(l,6)]-~-aGraelgood accep- maintaining structural integrity of the enzyme and must be tors for the B-galactosyltransferase from Ehcrleilclhs, suggesting that the enzymmeay participate in the biooccupied prior to of a second metal binding of any substrate
Summary
The transferase wassolubilized with the nonionic detergent, Lubrol PX, under conditions that yielded maximal recovery and stability of the enzyme This treatment resulted in the extraction of an a-galactosyltransferase from the microsomal membranes of Ehrlich cells. T o determine the intersugar glycosidic linkage of the Ehrlich cell p-galactosyltransferase reaction product, soluble GlcNAc (10 mM) and UDP-["CIGal (0.47mM,4.25 Ci/mol) were incubated in the presence of purified p-galactosyltransferase (5 ng) exactly as described above. The resulting preparation of P-galactosyltransferase from Ehrlich tumor cells was isolated in a 36%overall yield representing a 17,560-foldpurification (step 5 in Table I), and exhibited a specific activity similar to that reported for other 8-galactosyltransferases.The enzyme was stored at -20 "C in a neutralpH buffer containing manganese, detergent, and glycerol, without substantial loss of activity for at least 3 months. Storage in the absence of MnCL or Acceptor-binding Site ofP-Galactosyltransferase
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