The pyrophosphorylase that condenses UTP and GlcNAc-1-P was purified 9500-fold to near homogeneity from the soluble fraction of pig liver extracts. At the final stage of purification, the enzyme was quite stable and could be kept for at least 4 months in the freezer with only slight loss of activity. On native gels, the purified enzyme showed a single protein band, and this band was estimated to have a molecular mass of approximately125 kDa on Sephacryl S-300. SDS-polyacrylamide gel electrophoresis analysis of the enzyme gave three protein bands of 64, 57, and 49 kDa, but these polypeptides are all closely related based on the following. 1) All three polypeptides show strong cross-reactivity with antibody prepared against the 64-kDa band. 2) All three proteins become labeled with either the UDP-GlcNAc photoaffinity probe azido-125I-salicylate-allylamine-UDP-GlcNAc or a similar UDP-GalNAc photoaffinity probe, and either labeling was inhibited in a specific and concentration-dependent manner by unlabeled UDP-GlcNAc or UDP-GalNAc. Thus, the enzyme is probably a homodimer composed of two 64-kDa subunits. The purified enzyme had an unusual specificity in that, at higher substrate concentrations, it utilized UDP-GalNAc as a substrate as well as UDP-GlcNAc in the reverse direction and GalNAc-1-P as well as GlcNAc-1-P in the forward direction. However, the Km for the GalNAc substrates was considerably higher than that for GlcNAc derivatives. This activity for synthesizing UDP-GalNAc was not due to epimerase activity since no UDP-GalNAc could be detected when the enzyme was incubated with UDP-GlcNAc for various periods of time. The pyrophosphorylase required a divalent cation, with Mn2+ being best at 0.5-1 mM, and the pH optimum was between 8.5 and 8.9.

Using isopycnic sucrose gradients, we have ascertained the subcellular location of several enzymes involved in the processing of the N-linked oligosaccharides of glycoproteins in developing cotyledons of the common bean, Phaseolus vulgaris. All are localized in the endoplasmic reticulum (ER) or Golgi complex as determined by co-sedimentation with the ER marker, NADH-cytochrome c reductase, or the Golgi marker, glucan synthase I. Glucosidase activity, which removes glucose residues from Glc3Man,(GlcNAc)2, was found exclusively in the ER. All other processing enzymes, which act subsequent to the glucose trimming steps, are associated with the Golgi. These include mannosidase I (removes 1-2 mannose residues from Man6 4GlcNAcl2), mannosidase II (removes mannose residues from GlcNAcMans5GlcNAcI2), and fucosyltransferase (transfers a fucose residue to the Asn-linked GlcNAc of appropriate glycans). We have previously reported the localization of two other glycan modifying enzymes (GlcNAc-transferase and xylosyltransferase activities) in the Golgi complex. Attempts at subfractionation of the Golgi fraction on shallow sucrose gradients yielded similar patterns of distribution for all the Golgi processing enzymes. Subfractionation on Percoll gradients resulted in two peaks of the Golgi marker enzyme inosine diphosphatase, whereas the glycan processing enzymes were all enriched in the peak of lower density. These results do not lend support to the hypothesis that N-linked oligosaccharide processing enzymes are associated with Golgi cisternae of different densities.Recent evidence from several laboratories indicates that plant glycoproteins contain both high mannose and complex glycans attached to the polypeptide via a GlcNAc -. asparagine bond. The high mannose glycans have the general structure Man5-9 (GlcNAc)2; the complex glycans usually have fewer mannose residues, additional GlcNAc, and other sugars, such as fucose, xylose, and/or galactose (4). Glc3Man9(GlcNAc)2 to Man9(GlcNAc)2; a-mannosidase I, which removes up to four a 1-2 linked mannose residues from Mang(GlcNAc)2 to yield Man5-8(GlcNAc)2; and a-mannosidase II, which removes an a 1-3 and a 1-6 mannose from GlcNAc Man5(GlcNAc)2 to produce GlcNAcMan3(GlcNAc)2. The various glycosyltransferases transfer sugars (GlcNAc, fucose, xylose, and galactose) from their nucleoside diphosphate donors to specific N-linked glycan acceptors in a preferred sequence. GlcNAc-transferase I precedes while GlcNAc transferase II follows the action of a-mannosidase II to generate GlcNAcMan5(GlcNAc)2 and (GlcNAc)2Man3(GlcNAc)2, respectively. The latter product is an excellent acceptor for both fucosyl and xylosyltransferase (15), and presumably also for galactosyltransferase. Figure 1 shows the structure of the complex oligosaccharide that would result from the glycan-processing steps described above.The subcellular locations of several of these glycan-processing enzymes in plants have been ascertained indirectly through characterization of the oligosaccharides obtained from, and ...

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