Lentiginosine, a dihydroxyindolizidine alkaloid, was extracted from the leaves of Astragalus lentiginosus with hot methanol and was purified to homogeneity by ion-exchange, thin-layer, and radial chromatography. A second dihydroxyindolizidine, the 2-epimer of lentiginosine, was also purified to apparent homogeneity from these extracts. Gas chromatography of the two isomers (as the TMS derivatives) showed that they were better than 95% pure; lentiginosine eluted at 8.65 min and the 2-epimer at 9.00 min. Both compounds had a molecular ion in their mass spectra of 157, and the NMR spectra demonstrated that both were dihydroxyindolizidines differing in the configuration of the hydroxyl group at carbon 2. Lentiginosine was found to be a reasonably good inhibitor of the fungal alpha-glucosidase, amyloglucosidase (Ki = 1 x 10(-5) M), but it did not inhibit other alpha-glucosidases (i.e., sucrase, maltase, yeast alpha-glucosidase, glucosidase I) nor any other glycosidases. The 2-epimer had no activity against any of the glycosidases tested.

We show that Mycobacterium smegmatis has an enzyme catalyzing transfer of maltose from [ 14 C]maltose 1-phosphate to glycogen. This enzyme was purified 90-fold from crude extracts and characterized. Maltose transfer required addition of an acceptor. Liver, oyster, or mycobacterial glycogens were the best acceptors, whereas amylopectin had good activity, but amylose was a poor acceptor. Maltosaccharides inhibited the transfer of maltose from [14 C]maltose-1-P to glycogen because they were also acceptors of maltose, and they caused production of larger sized radioactive maltosaccharides. When maltotetraose was the acceptor, over 90% of the 14 C-labeled product was maltohexaose, and no radioactivity was in maltopentaose, demonstrating that maltose was transferred intact. Stoichiometry showed that 0.89 mol of inorganic phosphate was produced for each micromole of maltose transferred to glycogen, and 56% of the added maltose-1-P was transferred to glycogen. This enzyme has been named ␣1,4-glucan:maltose-1-P maltosyltransferase (GMPMT). Transfer of maltose to glycogen was inhibited by micromolar amounts of inorganic phosphate or arsenate but was only slightly inhibited by millimolar concentrations of glucose-1-P, glucose-6-P, or inorganic pyrophosphate. GMPMT was compared with glycogen phosphorylase (GP). GMPMT catalyzed transfer of [ 14 C]maltose-1-P, but not [ 14 C]glucose-1-P, to glycogen, whereas GP transferred radioactivity from glucose-1-P but not maltose-1-P. GMPMT and GP were both inhibited by 1,4-dideoxy-1,4-imino-D-arabinitol, but only GP was inhibited by isofagomine. Because mycobacteria that contain trehalose synthase accumulate large amounts of glycogen when grown in high concentrations of trehalose, we propose that trehalose synthase, maltokinase, and GMPMT represent a new pathway of glycogen synthesis using trehalose as the source of glucose.

Mannosidase II was purified from mung bean seedlings to apparent homogeneity by using a combination of techniques including DEAE-cellulose and hydroxyapatite chromatography, gel filtration, lectin affinity chromatography, and preparative gel electrophoresis. The release of radioactive mannose from GlcNAc[3H]Man5GlcNAc was linear with time and protein concentration with the purified protein, did not show any metal ion requirement, and had a pH optimum of 6.0. The purified enzyme showed a single band on SDS gels that migrated with the Mr 125K standard. The enzyme was very active on GlcNAcMan5GlcNAc but had no activity toward Man5GlcNAc, Man9GlcNAc, Glc3Man9GlcNAc, or other high-mannose oligosaccharides. It did show slight activity toward Man3GlcNAc. The first product of the reaction of enzyme with GlcNAcMan5GlcNAc, i.e., GlcNAcMan4GlcNAc, was isolated by gel filtration and subjected to digestion with endoglucosaminidase H to determine which mannose residue had been removed. This GlcNAcMan4GlcNAc was about 60% susceptible to Endo H indicating that the mannosidase II preferred to remove the alpha 1,6-linked mannose first, but 40% of the time removed the alpha 1,3-linked mannose first. The final product of the reaction, GlcNAcMan3GlcNAc, was characterized by gel filtration and various enzymatic digestions. Mannosidase II was very strongly inhibited by swainsonine and less strongly by 1,4-dideoxy-1,4-imino-D-mannitol. It was not inhibited by deoxymannojirimycin.

L-Fucokinase was purified to apparent homogeneity from pig kidney cytosol. The molecular mass of the enzyme on a gel filtration column was 440 kDa, whereas on SDS gels a single protein band of 110 kDa was observed. This 110-kDa protein was labeled in a concentration-dependent manner by azido-[ 32 P]ATP, and labeling was inhibited by cold ATP. The 110-kDa protein was subjected to endo-Lys-C digestion, and several peptides were sequenced. These showed very little similarity to other known protein sequences. The enzyme phosphorylated L-fucose using ATP to form ␤-L-fucose-1-P. Of many sugars tested, the only other sugar phosphorylated by the purified enzyme was D-arabinose, at about 10% the rate of L-fucose. Many of the properties of the enzyme were determined and are described in this paper. This enzyme is part of a salvage pathway for reutilization of L-fucose and is also a valuable biochemical tool to prepare activated L-fucose derivatives for fucosylation reactions. 6-Deoxy-L-galactose (L-fucose)1 is an important sugar in animal cells since it is involved in various recognition reactions of glycoproteins and glycolipids (1). Thus, oligosaccharides that have ␣-1,2-linked L-fucose are precursors for blood group A and B antigens (2). In the Lewis blood group antigens, Gal␤1,3 (Fuc␣1,4), GlcNAc-R, and Fuc␣1,2Gal␤1,3(Fuc␣1,4)GlcNAc-R are determinants for Lewis a and Lewis b blood group antigens. In addition, fucosylated and sialylated oligosaccharides have been found to be the recognition molecules for the E-and P-selectins, two members of the selectin family of cell adhesion molecules (3). These selectins and their fucosylated (and sialylated) ligands are important in inflammation and in the recognition of leukocytes for endothelial cells (4).The primary pathway for the formation of L-fucose in procaryotic and eucaryotic cells is from D-mannose via an internal oxidation reduction and then epimerization of GDP-D-mannose to produce GDP-L-fucose (5-8). However, studies in rats showed that radiolabeled L-fucose could be incorporated into glycoproteins (9, 10), suggesting an alternate route for activation of L-fucose. An L-fucokinase that synthesizes ␤-L-fucose-1-phosphate (11) and a GDP-L-fucose pyrophosphorylase (12) were partially purified from pig liver. However, the fucokinase preparation had rather broad substrate specificity with regard to sugar and nucleoside triphosphate, probably because of contaminating enzymes such as hexokinase in the partially purified fraction. In the present report, we describe the purification to apparent homogeneity of the pig kidney fucokinase. This enzyme preparation was very specific for L-fucose, and the only other sugar that could be phosphorylated, at about 10% of the rate with L-fucose, was D-arabinose. The fucokinase is also quite specific for ATP as the phosphate donor. This enzyme should be valuable for the synthesis of large amounts of L-fucose-1-P, as well as for the formation of radiolabeled fucose-1-P. EXPERIMENTAL PROCEDURES Materials L-[3 H]Fucose (52 Ci/mmol) and other ...

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.