Identification and modification of the uridine-binding site of the UDP-GalNAc (GlcNAc) pyrophosphorylase.

UDP-GalNAc pyrophosphorylase (UDP-GalNAcPP; AGX1) catalyzes the synthesis of UDP-GalNAc from UTP and GalNAc-1-P. The 475-amino acid protein (57 kDa protein) also synthesizes UDP-GlcNAc at about 25% the rate of UDP-GalNAc. The cDNA for this enzyme, termed AGX1, was cloned in Escherichia coli, and expressed as an active enzyme that cross-reacted with antiserum against the original pig liver UDP-HexNAcPP. In the present study, we incubated recombinant AGX1 with N(3)-UDP-[(32)P]GlcNAc and N(3)-UDP-[(32)P]GalNAc probes to label the nucleotide-binding site. Proteolytic digestions of the labeled enzyme and analysis of the resulting peptides indicated that both photoprobes cross-linked to one 24-amino acid peptide located between residues Val(216) and Glu(240). Four amino acids in this peptide were found to be highly conserved among closely related enzymes, and each of these was individually modified to alanine. Mutation of Gly(222) to Ala in the peptide almost completely eliminated UDP-GlcNAc and UDP-GalNAc synthesis, while mutation of Gly(224) to Ala, almost completely eliminated UDP-GalNAc synthesis, but UDP-GlcNAc was only diminished by 50%. Both of these mutations also resulted in almost complete loss of the ability of the mutated proteins to cross-link N(3)-UDP-[(32)P]GlcNAc or N(3)-UDP-[(32)P]GalNAc. On the other hand, mutations of either Pro(220) or Tyr(227) to Ala did not greatly affect enzymatic activity, although there was some reduction in the ability of these proteins to cross-link the photoaffinity probes. We also mutated Gly(111) to Ala since this amino acid was reported to be necessary for catalysis (Mio, T., Yabe, T., Arisawa, M., and Yamada-Okabe, H. (1998) J. Biol. Chem. 273, 14392-14397). The Gly(111) to Ala mutant lost all enzymatic activity, but interestingly enough, this mutant protein still cross-linked the radioactive N(3)-UDP-GlcNAc although not nearly as well as the wild type. On the other hand, mutation of Arg(115) to Ala had no affect on enzymatic activity although it also reduced the amount of cross-linking of N(3)-UDP-[(32)P]GlcNAc. These studies help to define essential amino acids at or near the nucleotide-binding site and the catalytic site, as well as peptides involved in binding and catalysis.

GDP-L-fucose pyrophosphorylase. Purification, cDNA cloning, and properties of the enzyme.

The enzyme that catalyzes the formation of GDP-L-fucose from GTP and beta-L-fucose-1-phosphate (i.e. GDP-beta-L-fucose pyrophosphorylase, GFPP) was purified about 560-fold from the cytosolic fraction of pig kidney. At this stage, there were still a number of protein bands on SDS gels, but only the 61-kDa band became specifically labeled with the photoaffinity substrate, azido-GDP-L-[32P]fucose. Several peptides from this 61-kDa band were sequenced and these sequences were used for cloning the gene. The cDNA clone yielded high levels of GFPP activity when expressed in myeloma cells and in a baculovirus system, demonstrating that the 61-kDa band is the authentic GFPP. The porcine tissue with highest specific activity for GFPP was kidney, with lung, liver, and pancreas being somewhat lower. GFPP was also found in Chinese hamster ovary, but not Madin-Darby canine kidney cells. Northern analysis showed the mRNA in human spleen, prostate, testis, ovary, small intestine, and colon. GFPP was stable at 4 (o)C in buffer containing 50 mM sucrose, with little loss of activity over a 9-day period. GTP was the best nucleoside triphosphate substrate but significant activity was also observed with ITP and to a lesser extent with ATP. The enzyme was reasonably specific for beta-L-fucose-1-P, but could also utilize alpha-D-arabinose-1-P to produce GDP-alpha-D-arabinose. The product of the reaction with GTP and alpha-L-fucose-1-P was characterized as GDP-beta-L-fucose by a variety of chemical and chromatographic methods.

A 17-amino acid insert changes UDP-N-acetylhexosamine pyrophosphorylase specificity from UDP-GalNAc to UDP-GlcNAc.

We previously reported the purification of a UDP-N-acetylhexosamine (UDP-HexNAc) pyrophosphorylase from pig liver that catalyzed the synthesis of both UDP-GlcNAc and UDP-GalNAc from UTP and the appropriate HexNAc-1-P (Szumilo, T., Zeng, Y., Pastuszak, I., Drake, R., Szumilo, H., and Elbein, A. D. (1996) J. Biol. Chem. 271, 13147-13154). Both sugar nucleotides were synthesized at nearly the same rate, although the Km for GalNAc-1-P was about 3 times higher than for GlcNAc-1-P. Based on native gels and SDS-polyacrylamide gel electrophoresis, the enzyme appeared to be a dimer of 120 kDa composed of two subunits of about 57 and 64 kDa. Three peptides sequenced from the 64-kDa protein and two from the 57-kDa protein showed 100% identity to AGX1, a 57-kDa protein of unknown function from human sperm. An isoform called AGX2 is identical in sequence to AGX1 except that it has a 17-amino acid insert near the carboxyl terminus. We expressed the AGX1 and AGX2 genes in Escherichia coli. The protein isolated from the AGX1 clone comigrated on SDS gels with the liver 57-kDa pyrophosphorylase subunit and was 2-3 times more active with GalNAc-1-P than with GlcNAc-1-P. On the other hand, the protein from the AGX2 clone migrated with the liver 64-kDa pyrophosphorylase subunit and had 8-fold better activity with GlcNAc-1-P than with GalNAc-1-P. These results indicate that insertion of the 17-amino acid peptide modifies the specificity of the pyrophosphorylase from synthesis of UDP-GalNAc to synthesis of UDP-GlcNAc.

Synthesis of 5-azido-UDP-N-acetylhexosamine photoaffinity analogs and radiolabeled UDP-N-acetylhexosamines.

Nuleotide sugar photoaffinity analogs have proven to be useful in the identification and characterization of glycosyltransferases. A radioenzymatic synthesis of [32P]5-azido-UDP-N-acetylglucosamine has been accomplished using 5-azido-UTP, [gamma-32P]ATP, porcine N-acetylgalactosamine kinase, and Escherichia coli UDP-N-acetylglucosamine pyrophosphorylase, GlmU. This general enzymatic scheme was useful for the synthesis of [32P]5-azido-UDP-N-acetylgalactosamine and high-specific-activity [3H] or [32P]UDP-N-acetylhexosamines. A new chemical synthesis method for generating 5-azido-uridine compounds was also developed. [32P]5-Azido-UDP-N-acetylglucosamine was functionally characterized using different soluble and membrane-associated glycosyltransferases which utilize UDP-GlcNAc as a substrate. Site-specific photoincorporation was observed for partially purified GlmU and porcine UDP-GlcNAc pyrophosphorylase. The photoprobe also effectively photoincorporated into the alpha- and beta-subunits of purified bovine UDP-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase. Lastly, the photoprobe was also effective at photolabeling Streptococcus pyogenes hyaluronate synthase in membrane preparations.

Purification to apparent homogeneity and properties of pig kidney L-fucose kinase.

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-[32P]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 beta-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.

Synthesis and utilization of GDP-D-arabinopyranoside.

We describe a procedure for the enzymatic synthesis of labeled or unlabeled GDP-D-arabinopyranoside. This method uses two enzymes purified from pig kidney: an L-fucokinase and a GDP-L-fucose pyrophosphorylase. The isolated GDP-D-[3H]arabinose served as a precursor for arabinose addition to lipophosphoglycan (LPG) of Leishmania major, using a parasite membrane fraction as the source of arabinosyltransferase. The procedures described provide a useful means for obtaining radiolabeled GDP-D-arabinopyranoside to study synthesis of D-arabinopyranoside-containing glycoconjugates.

Identification of the GalNAc kinase amino acid sequence.

A new kinase that forms GalNAc-1-P was purified from pig kidney cytosol and identified on gels by labeling with N3-[32P]ATP (Pastuszak, I., Drake, R., and Elbein, A. D. (1996) J. Biol. Chem. 271, in press). A 50-kDa labeled protein was eluted, digested with trypsin, and the sequences of four peptides representing 49 amino acids showed 90% identity to sequence of human galactokinase reported to be on chromosome 15. To resolve this dilemma, activities and substrate specificities of galactokinase and GalNAc kinase from human and pig kidney, as well as of galactokinase from the yeast clone transfected with the cDNA from presumptive human galactokinase, were compared. The purified galactokinases phosphorylated galactose, but not GalNAc, whereas GalNAc kinase also phosphorylated galactose when this sugar was present at millimolar concentrations. Extracts of gal 1(-) yeast clone, transfected with presumptive human galactokinase cDNA, had very low galactokinase activity even when yeast were grown on galactose, but good activity with GalNAc. On the other hand, the wild type yeast phosphorylated galactose, but not GalNAc. These data indicate that the sequence reported for galactokinase on chromosome 15 is that of GalNAc kinase, which can phosphorylate galactose when this sugar is present at millimolar concentrations. This transfection thus allows the yeast mutant to grow slowly on galactose-containing media.

Kidney N-acetylgalactosamine (GalNAc)-1-phosphate kinase, a new pathway of GalNAc activation.

A new enzyme that phosphorylates GalNAc at position 1 to form GalNAc-alpha-1P was purified approximately 1275-fold from the cytosolic fraction of pig kidney, and the properties of the enzyme were determined. The kinase is quite specific for GalNAc as the phosphate acceptor and is inactive with GlcNAc, ManNAc, glucose, galactose, mannose, GalN, and GlcN. This enzyme is clearly separated from galactokinase by chromatography on phenyl-Sepharose. The GalNAc kinase has a pH optimum between 8.5 and 9.0 and requires a divalent cation in the order Mg2+ > Mn2+ > Co2+, with optimum Mg2+ concentration at approximately 5 mM. The enzyme was most active with ATP as the phosphate donor, but slight activity was observed with ITP, acetyl-P, and phosphoenolpyruvate. Enzyme activity was highest in porcine and human kidney and porcine liver, but was low in most other tissues. Cultured HT-29 cells also had high activity for this kinase. The purified enzyme fraction was incubated with azido-[32P]ATP, exposed to UV light, and run on SDS gels. A 50-kDa protein was labeled, and this labeling showed saturation kinetics with increasing amounts of the probe and was inhibited by unlabeled ATP. Although the most purified GalNAc kinase preparation still had two bands that labeled with ATP, maximum labeling of the 50-kDa protein, but not the 66-kDa band, was coincident with maximum GalNAc kinase activity on a column of DEAE-Cibacron blue. On Sephacryl S-300, the native enzyme has a molecular mass of 48-51 kDa, indicating that the active kinase is a monomer. The product of the reaction was characterized as GalNAc-alpha-1-P by various chemical procedures.

Synthesis of aryl azide derivatives of UDP-GlcNAc and UDP-GalNAc and their use for the affinity labeling of glycosyltransferases and the UDP-HexNAc pyrophosphorylase.

The chemical synthesis and utilization of two photoaffinity analogs, 125I-labeled 5-[3-(p-azidosalicylamido)-1-propenyl]-UDP-GlcNAc and -UDP-GalNAc, is described. Starting with either UDP-GlcNAc or UDP-GalNAc, the synthesis involved the preparation of the 5-mercuri-UDP-HexNAc and then attachment of an allylamine to the 5 position to give 5-(3-amino)allyl-UDP-HexNAc. This was followed by acylation with N-hydroxysuccinimide p-aminosalicylic acid to form the final product, i.e., 5-[3-(p-azidosalicylamido)-1-propenyl]-UDP-GlcNAc or UDP-GalNAc. These products could then be iodinated with chloramine T to give the 125I-derivatives. Both the UDP-GlcNAc and the UDP-GalNAc derivatives reacted in a concentration-dependent manner with a highly purified UDP-HexNAc pyrophosphorylase, and both specifically labeled the subunit(s) of this protein. The labeling of the protein by the UDP-GlcNAc derivative was inhibited in dose-dependent fashion by either unlabeled UDP-GlcNAc or unlabeled UDP-GalNAc. Likewise, labeling with the UDP-GalNAc probe was blocked by either UDP-GlcNAc or UDP-GalNAc. The UDP-GlcNAc probe also specifically labeled a partially purified preparation of GlcNAc transferase I.

Purification to homogeneity and properties of UDP-GlcNAc (GalNAc) pyrophosphorylase.

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.

Partial Purification, Photoaffinity Labeling, and Properties of Mung Bean UDP-Glucose:Dolicholphosphate Glucosyltransferase.

UDP-glucose:dolichylphosphate glucosyltransferase has been purified 734-fold from Triton X-100 solubilized mung bean (Phaseolus aureus) microsomes. The partially purified enzyme has broad pH optima of activity from 6.0 to 7.0 and is maximally stimulated with 10 millimolar MgCl(2). The K(m) for UDP-glucose was determined as 27 micromolar, and the K(m) for dolichol-P was 2 micromolar. Using the UDP-glucose photoaffinity analog, 5-azido-UDP-glucose, a polypeptide of 39 kilodaltons on sodium dodecyl sulfate-polyacrylamide gels was identified as the catalytic subunit of the enzyme. Photoinsertion into this 39-kilodalton polypeptide with [(32)P]5-azido-UDP-glucose was saturable, and was maximally protected with the native substrate UDP-glucose. 5-Azido-UDP-glucose behaves competitively with UDP-glucose in enzyme assays, and upon photolysis inhibits activity in proportion to its concentration. This study represents the first subunit identification of a plant glycosyltransferase involved in the biosynthesis of the lipid-linked oligosaccharides that are precursors of N-linked glycoproteins.

Mannostatin A, a new glycoprotein-processing inhibitor.

Mannostatin A is a metabolite produced by the microorganism Streptoverticillium verticillus and reported to be a potent competitive inhibitor of rat epididymal alpha-mannosidase. When tested against a number of other arylglycosidases, mannostatin A was inactive toward alpha- and beta-glucosidase and galactosidase as well as beta-mannosidase, but it was a potent inhibitor of jack bean, mung bean, and rat liver lysosomal alpha-mannosidases, with estimated IC50's of 70 nM, 450 nM, and 160 nM, respectively. The type of inhibition was competitive in nature. This compound also proved to be an effective competitive inhibitor of the glycoprotein-processing enzyme mannosidase II (IC50 of about 10-15 nM with p-nitrophenyl alpha-D-mannopyranoside as substrate, and about 90 nM with [3H]mannose-labeled GlcNAc-Man5GlcNAc as substrate). However, it was virtually inactive toward mannosidase I. The N-acetylated derivative of mannostatin A had no inhibitory activity. In cell culture studies, mannostatin A also proved to be a potent inhibitor of glycoprotein processing. Thus, in influenza virus infected Madin Darby canine kidney (MDCK) cells, mannostatin A blocked the normal formation of complex types of oligosaccharides on the viral glycoproteins and caused the accumulation of hybrid types of oligosaccharides. This observation is in keeping with other data which indicate that the site of action of mannostatin A is mannosidase II. Thus, mannostatin A represents the first nonalkaloidal processing inhibitor and adds to the growing list of chemical structures that can have important biological activity.

Purification and properties of arylmannosidases from mung bean seedlings and soybean cells.

Two arylmannosidases (signified as A and B) were purified to homogeneity from soluble and microsomal fractions of mung bean seedlings. Arylmannosidase A from the microsomes appeared the same on native gels and on SDS gels as soluble arylmannosidase A, the same was true for arylmannosidase B. Sedimentation velocity studies indicated that both enzymes were homogeneous, and that arylmannosidase A had a molecular mass of 237 kd while B had a molecular mass of 243 kd. Arylmannosidase A showed two major protein bands on SDS gels with molecular masses of 60 and 55 kd, and minor bands of 79, 39 and 35 kd. All of these bands were N-linked since they were susceptible to digestion by endoglucosaminidase H. In addition, at least the major bands could be detected by Western blots with antibody raised against the xylose moiety of N-linked plant oligosaccharides, and they could also be labeled in soybean suspension cells with [2-3H]mannose. Arylmannosidase B showed three major bands with molecular masses of 72, 55 and 45 kd, and minor bands of 42 and 39 kd. With the possible exception of the 45 and 42 kd bands, all of these bands are glycoproteins. Arylmannosidases A and B showed somewhat different kinetics in terms of mannose release from high-mannose oligosaccharides, but they were equally susceptible to inhibition by swainsonine and mannostatin A. Polyclonal antibody raised against the arylmannosidase B cross-reacted equally well with arylmannosidase A from mung bean seedlings and with arylmannosidase from soybean cells. However, monoclonal antibody against mung bean arylmannosidase A was much less effective against arylmannosidase B. Antibody was used to examine the biosynthesis and structure of the carbohydrate chains of arylmannosidase in soybean cells grown in [2-3H]mannose. Treatment of the purified enzyme with Endo H released approximately 50% of the radioactivity, and these labeled oligosaccharides were of the high-mannose type, i.e. mostly Man9GlcNAc. The precipitated protein isolated from the Endo H treatment still contained 50% of the radioactivity, and this was present in modified structures that probably contain xylose residues.

Purification to homogeneity and properties of glucosidase II from mung bean seedlings and suspension-cultured soybean cells.

Glucosidase II was purified approximately 1700-fold to homogeneity from Triton X-100 extracts of mung bean microsomes. A single band with a molecular mass of 110 kDa was seen on sodium dodecyl sulfate gels. This band was susceptible to digestion by endoglucosaminidase H or peptide glycosidase F, and the change in mobility of the treated protein indicated the loss of one or two oligosaccharide chains. By gel filtration, the native enzyme was estimated to have a molecular mass of about 220 kDa, suggesting it was composed of two identical subunits. Glucosidase II showed a broad pH optima between 6.8 and 7.5 with reasonable activity even at 8.5, but there was almost no activity below pH 6.0. The purified enzyme could use p-nitrophenyl-alpha-D-glucopyranoside as a substrate but was also active with a number of glucose-containing high-mannose oligosaccharides. Glc2Man9GlcNAc was the best substrate while activity was significantly reduced when several mannose residues were removed, i.e. Glc2Man7-GlcNAc. The rate of activity was lowest with Glc1Man9GlcNAc, demonstrating that the innermost glucose is released the slowest. Evidence that the enzyme is specific for alpha 1,3-glucosidic linkages is shown by the fact that its activity on Glc2Man9GlcNAc was inhibited by nigerose, an alpha 1,3-linked glucose disaccharide, but not by alpha 1,2 (kojibiose)-, alpha 1,4(maltose)-, or alpha 1,6 (isomaltose)-linked glucose disaccharides. Glucosidase II was strongly inhibited by the glucosidase processing inhibitors deoxynojirimycin and 2,6-dideoxy-2,6-imino-7-O-(beta-D- glucopyranosyl)-D-glycero-L-guloheptitol, but less strongly by castanospermine and not at all by australine. Polyclonal antibodies prepared against the mung bean glucosidase II reacted with a 95-kDa protein from suspension-cultured soybean cells that also showed glucosidase II activity. Soybean cells were labeled with either [2-3H]mannose or [6-3H]galactose, and the glucosidase II was isolated by immunoprecipitation. Essentially all of the radioactive mannose was released from the protein by treatment with endoglucosaminidase H. The labeled oligosaccharide(s) released by endoglucosaminidase H was isolated and characterized by gel filtration and by treatment with various enzymes. The major oligosaccharide chain on the soybean glucosidase II appeared to be a Man9(GlcNAc)2 with small amounts of Glc1Man9(GlcNAc)2.

Purification to homogeneity and properties of mannosidase II from mung bean seedlings.

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.

Lentiginosine, a dihydroxyindolizidine alkaloid that inhibits amyloglucosidase.

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.

Plant glucosidase II catalyzes a transglucosylation reaction in addition to the hydrolytic reaction.

When the purified plant glucosidase II was incubated with [3H]Glc2Man9GlcNAc in the presence of glycerol and the products were analyzed by gel filtration, a large peak of radioactivity emerged just before the glucose standard. The formation of this peak was dependent on both the presence of Glc2Man9GlcNAc and the presence of glycerol, and the amount of this product increased with time of incubation and amount of glucosidase II in the incubation. When the incubation was performed with [3H]Glc2Man9GlcNAc plus [14C]glycerol, the product contained both 14C and 3H. Strong acid hydrolysis of the purified product gave rise to [14C]glycerol and [3H]glucose. Various other chemical treatments and chromatographic techniques showed that the product was glucosyl----glycerol. Since the glucose was released by alpha-glucosidase, the product must be glucosyl-alpha-glycerol. This study demonstrates that the processing glucosidase II catalyzes a trans-glycosylation reaction in the presence of acceptors like glycerol. Since this transglycosylation reaction may give rise to unexpected products, investigators should be aware of its possible occurrence.

Occurrence of a large molecular size form of polyphosphate-glucose phosphotransferase in extracts of Mycobacterium tuberculosis H37Ra.

Extracts prepared by sonicating Mycobacterium tuberculosis H37Ra cells with subsequent centrifugation at 18,000 X g proved to contain a very large molecular size form of polyphosphate-glucose phosphotransferase. The enzyme was separable by polyacrylamide gel electrophoresis, DEAE-cellulose chromatography or ultracentrifugation. When rechromatographed at alkaline pH values, it gave rise to one of the soluble forms of lower molecular weight. The conversion also took place as a result of n-butanol extraction or salting out with ammonium sulfate and heating of dissolved pellet. Under certain conditions the lower-molecular weight enzyme converted to the higher-molecular weight form by association with a hitherto undefined cell constituent. It is assumed that both ionic and hydrophobic forces play a role in this interconversion phenomenon.

A mannoglucokinese of Mycobacterium tuberculosis H37Ra.

An ATP: D-glucose and D-mannose 6-phosphotransferase activity was found in Mycobacterium tuberculosis HERa. The activity was separated from other ATP- and polyphosphate D-glucose phosphotransferases in a procedure involving precipitation with ammonium sulfate, treatment with calcium phosphate gel, DEAE-cellulose and DEAE-Sephadex A50 chromatography. The optimum pH of the phosphorylation reaction was from 9 to 10.5. The hexokinase phosphorylated D-glucose with a Km of 20 mM under conditions of MgATP saturation. The Km for MgATP was 0.2 mM. The enzyme showed a higher activity on D-mannose at a saturation level being about 100-fold lower than that of D-glucose; it did not utilize either D-fructose or D-glucosamine. Inorganic poly(P) could not replace ATP as the phosphate donor. M. tuberculosis H37Ra was unable to grow on D-mannose which may suggest that the enzyme studied is involved in endogenous metabolism of this sugar.