Trehalose-phosphate synthase of Mycobacterium tuberculosis. Cloning, expression and properties of the recombinant enzyme.

The trehalose-phosphate synthase (TPS) of Mycobacterium smegmatis was previously purified to apparent homogeneity and several peptides from the 58 kDa protein were sequenced. Based on that sequence information, the gene for TPS was identified in the Mycobacterium tuberculosis genome, and the gene was cloned and expressed in Escherichia coli with a (His)6 tag at the amino terminus. The TPS was expressed in good yield and as active enzyme, and was purified on a metal ion column to give a single band of approximately 58 kDa on SDS/PAGE. Approximately 1.3 mg of purified TPS were obtained from a 1-L culture of E. coli ( approximately 2.3 g cell paste). The purified recombinant enzyme showed a single band of approximately 58 kDa on SDS/PAGE, but a molecular mass of approximately 220 kDa by gel filtration, indicating that the active TPS is probably a tetrameric protein. Like the enzyme originally purified from M. smegmatis, the recombinant enzyme is an unusual glycosyltransferase as it can utilize any of the nucleoside diphosphate glucose derivatives as glucosyl donors, i.e. ADP-glucose, CDP-glucose, GDP-glucose, TDP-glucose and UDP-glucose, with ADP-glucose, GDP-glucose and UDP-glucose being the preferred substrates. These studies prove conclusively that the mycobacterial TPS is indeed responsible for catalyzing the synthesis of trehalose-P from any of the nucleoside diphosphate glucose derivatives. Although the original enzyme from M. smegmatis was greatly stimulated in its utilization of UDP-glucose by polyanions such as heparin, the recombinant enzyme was stimulated only modestly by heparin. The Km for UDP-glucose as the glucosyl donor was approximately 18 mm, and that for GDP-glucose was approximately 16 mm. The enzyme was specific for glucose-6-P as the glucosyl acceptor, and the Km for this substrate was approximately 7 mm when UDP-glucose was the glucosyl donor and approximately 4 mm with GDP-glucose. TPS did not show an absolute requirement for divalent cations, but activity was increased about twofold by 10 mm Mn2+. This recombinant system will be useful for obtaining sufficient amounts of protein for structural studies. TPS should be a valuable target site for chemotherapeutic intervention in tuberculosis.

Differential and region-specific activation of mitogen-activated protein kinases following chronic administration of phencyclidine in rat brain.

We have previously demonstrated elevation of the extracellular signal-regulated kinase (ERK) pathway in the cerebellum from patients with schizophrenia, an illness that may involve dysfunction of the N-methyl-D-aspartate (NMDA) receptor. Since the NMDA antagonist, phencyclidine (PCP), produces schizophrenic-like symptoms in humans, and abnormal behavior in animals, we examined the effects of chronic PCP administration in time- and dose-dependent manner on ERK and two other members of mitogen-activated protein kinase family, c-Jun N-terminal protein kinase (JNK) and p38, in rat brain. Osmotic pumps for PCP (18 mg/kg/day) and saline (controls) were implanted subcutaneously in rats for three, 10, and 20 days. Using Western blot analysis, we found no change at three days, but a significant increase in the phosphorylation of ERK1, ERK2 and MEK in the cerebellum at 10- and 20-days of continuous PCP infusion. For the experiments involving various doses of PCP, rats were infused with PCP at concentrations of 2.5, 10, 18, or 25 mg/kg/day, or saline for 10 days. We observed a dose-dependent elevation in the phosphorylation of ERK1 and ERK2 only in the cerebellum but not in brainstem, frontal cortex or hippocampus. The activities of JNK and p38 were unchanged in all investigated brain regions including cerebellum. These results demonstrate that chronic infusion of PCP in rats produces a differential and brain region-specific activation of MAP kinases, suggesting a role for the ERK signaling pathway in PCP abuse and perhaps in schizophrenia.

Cloning, expression and characterization of the pig liver GDP-mannose pyrophosphorylase. Evidence that GDP-mannose and GDP-Glc pyrophosphorylases are different proteins.

GDP-Man, the mannosyl donor for most Man-containing polymers is formed by the transfer of Man-1-P to GTP to form GDP-Man and PPi. This reaction is catalyzed by the widespread and essential enzyme, GDP-Man pyrophosphorylase (GMPP). The pig liver GMPP consists of an alpha subunit (43 kDa) and a beta subunit (37 kDa). Purified pig GMPP catalyzes the synthesis of GDP-Glc (from Glc-1-P and GTP) and GDP-Man (from Man-1-P and GTP), but has higher activity for the formation of GDP-Glc than for synthesis of GDP-Man. In the present study, we report the cloning of the cDNA for the beta subunit of GMPP, and its expression in a bacterial system resulting in the formation of active enzyme. The full length cDNA encoding the beta subunit was isolated from a porcine cDNA library, and its predicted gene product showed high amino-acid sequence homology to GMPPs from other species. The gene was expressed in Escherichia coli cells, and a 37-kDa protein was over-produced in these cells. This gene product reacted strongly with antibody reactive to the native beta subunit of pig GMPP. Most interestingly, this recombinant protein had high activity for synthesizing GDP-Man (from Man-1-P and GTP), but very low activity for the formation of GDP-Glc (from Glc-1-P and GTP). Other properties of the recombinant protein were also analyzed. This study suggests that the beta subunit is the GMPP, whereas the alpha subunit, or a combination of both subunits, may have the GDP-Glc pyrophosphorylase activity.

Increased levels of transcription factors Elk-1, cyclic adenosine monophosphate response element-binding protein, and activating transcription factor 2 in the cerebellar vermis of schizophrenic patients.

BACKGROUND: We investigated the levels of transcription factors associated with activation of the mitogen-activated protein (MAP) kinase pathway in schizophrenics using postmortem brain samples. These studies were done to determine whether our previous findings of abnormal levels of the MAP kinases in the cerebellar vermis were linked to additional downstream targets of this signal transduction pathway. METHOD: We measured the protein levels of 3 transcription factors in nuclear fractions of postmortem samples from cerebellar vermis of 10 patients with schizophrenia and 13 control subjects: Elk-1, cyclic adenosine monophosphate (cAMP) response element binding protein (CREB), and activating transcription factor 2 (ATF-2). Studies in rats examined the postmortem stability and effect of haloperidol and risperidone on levels of Elk-1, cAMP, and ATF-2 proteins. RESULTS: We found a significant increase in the protein levels of Elk-1 (mean+SD, 4489+/-659 vs 2915+/-583 arbitrary densitometric units [P<.001]), CREB (mean +/- SD, 2149 1061 vs 904+/-711 arbitrary densitometric units [P=.003]) and ATF-2 (mean+/-SD, 1421 854 vs 512+/-394 arbitrary densitometric units [P=.003]) in the cerebellar vermis of schizophrenic subjects. Complementary studies in rats indicate that these findings can not be attributed to subacute treatment with antipsychotic medications. CONCLUSION: Taken together with the alterations of MAP kinases previously reported, and the findings of elevations of downstream transcription targets, we suggest that the MAP kinase signal transduction pathway contributes to the cerebellar abnormalities in schizophrenia.

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.

Mitogen-activated protein kinases in schizophrenia.

BACKGROUND: Mitogen-activated protein kinases (MAPKs) are important mediators of signal transduction from the cell surface to the nucleus and have been implicated in the integration of a variety of physiologic processes in most cells, including neurons. To investigate the possible involvement of MAPKs in schizophrenia, we compared the levels of the MAPK intermediates in postmortem brain tissue obtained from schizophrenic and control subjects. Our focus was on the cerebellar vermis because of evidence suggesting that schizophrenia is associated with abnormalities of structure, function, and signal transduction in this brain region. METHODS: Cytosolic proteins were fractionated by gel electrophoresis and subjected to Western blot analysis using polyclonal MAPK antibody, which detects total extracellular signal-regulated kinases (ERKs) 1 and 2 levels, and monoclonal MAP kinase phosphatase (MKP) 2 antibody. RESULTS: Schizophrenic subjects had increased levels of ERK2 [2763 +/- (SD) 203 vs. 2286 +/- 607 arbitrary units, U = 17, p < .05] in cerebellar vermis. The levels of a dual specificity tyrosine phosphatase, MKP2, were significantly decreased in cerebellar vermis (1716 +/- 465 versus 2372 +/- 429 arbitrary units, U = 12, p < .02) from schizophrenic patients. ERK1/MKP2 and ERK2/MKP2 ratios in cerebellar vermis, but not in other brain regions, were significantly different in schizophrenic subjects as compared to control subjects (U = 15, p < or = .027; U = 3, p < .001, respectively). CONCLUSIONS: MAPK levels are elevated in the cerebellar vermis of schizophrenic subjects. This could result from a protein dephosphorylation defect in vivo and might be involved in the pathology of the disease.

Purification and properties of mycobacterial GDP-mannose pyrophosphorylase.

The enzyme that catalyzes the formation of GDP-d-mannose from GTP and alpha-d-mannose-1-P was purified about 2300-fold to near homogeneity from the soluble fraction of Mycobacterium smegmatis. At the final stage of purification, a major protein band of 37 kDa was observed and this band was specifically labeled, and in a concentration-dependent manner, by the photoaffinity probe 8-N3-GDP[32P]-d-mannose. The purified enzyme was stable for several months when kept in the frozen state. The 37-kDa band was subjected to protein sequencing and one peptide sequence of 25 amino acids showed over 80% identity to GDP-mannose pyrophosphorylases of pig liver and Saccharomyces cerevesiae. In contrast to some other bacterial GDP-mannose pyrophosphorylases, the mycobacterial enzyme was not multifunctional and did not have phosphomannose isomerase or phosphoglucose isomerase activity. Also, in contrast to the pig liver enzyme which uses mannose-1-P or glucose-1-P plus GTP to synthesize either GDP-mannose or GDP-glucose, the mycobacterial enzyme was specific for mannose-1-P as the sugar phosphate substrate. The enzyme was also relatively specific for GTP as the nucleoside triphosphate substrate. ITP was about 18% as effective as GTP, but ATP, CTP, and UTP were inactive. The activity of the enzyme was inhibited by GDP-glucose and glucose-1-P, although neither was a substrate for this enzyme. The pH optimum for the enzyme was 8.0, and Mg2+ was the best cation with optimum activity at about 5 mM. This enzyme is important for producing the activated form of mannose for formation of cell wall lipoarabinomannan and various mannose-containing glycolipids and polysaccharides.

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.

Purification to homogeneity and properties of plant glucosidase I.

Glucosidase I was purified about 3600-fold to apparent homogeneity from the microsomal fraction of mung bean seedlings. The purified enzyme removed the terminal alpha1,2-linked glucose from Glc3Man9GlcNAc2-peptide or the endoglucosaminidase H (Endo H)-released oligosaccharide. Glucosidase I activity was inhibited by kojibiose [Glc(alpha1-2)Glc], but not by other glucose disaccharides. Removal of up to four mannose residues from the N-linked oligosaccharide had little effect on its utilization as a substrate for glucosidase I. The enzyme had a subunit molecular weight of 97 kDa on SDS gels and this was shifted to 94 kDa after treatment with Endo H or Endo F, suggesting that glucosidase I is an N-glycoprotein having one oligomannose-type oligosaccharide. Amino acid sequences of this enzyme showed considerable identity to the enzyme cloned from a human hippocampus cDNA library. The enzyme was inhibited by castanospermine, deoxynojirimycin, MDL, and trehazolin, but not by australine or kifunensine. On the other hand, the other processing glucosidase, glucosidase II, is sensitive to inhibition by australine, but not by trehazolin. Thus, these two inhibitors are useful to distinguish glucosidase I from glucosidase II. The mung bean glucosidase I is quite sensitive to the histidine modifying reagent diethyl pyrocarbonate, whereas the pig liver glucosidase I is not. On the other hand, pig liver and pig brain glucosidase I preparations are sensitive to the sulfhydryl reagent NEM (N-ethylmaleimide), whereas the plant enzyme is not. These sensitivities to amino acid modifiers suggest significant differences between the plant and animal glucosidase I, in terms of catalytic site or protein conformation.

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.

Specificity of the high-mannose recognition site between Enterobacter cloacae pili adhesin and HT-29 cell membranes.

Enterobacter cloacae has been implicated as one of the causative agents in neonatal infection and causes a septicemia thought to be initiated via the gastrointestinal tract. The adhesion of radiolabeled E. cloacae to HT-29 cells was concentration and temperature dependent and was effectively blocked by unlabeled bacteria or by millimolar concentrations of alpha-mannosides and micromolar concentrations of high-mannose oligosaccharides. A variety of well-characterized mannose oligosaccharides were tested as inhibitors of adhesion. The best inhibitor was the Man9(GlcNAc)2-tyrosinamide, which was considerably better than other tyrosinamide-linked oligosaccharides such as Man7(GlcNAc)2, Man6(GlcNAc)2 or Man5(GlcNAc)2. Further evidence that the bacteria preferred Man9(GlcNAc)2 structures was obtained by growing HT-29 cells in the presence of glycoprotein processing inhibitors that block mannosidase I and increase the amount of protein-bound Man9(GlcNAc)2 at the cell surface. Such cells bound 1.5- to 2-fold more bacteria than did control cells. The adhesin involved in binding to high-mannose structures was purified from isolated pili. On sodium dodecyl sulfate-gels, a 35-kDa protein was identified by its specific binding to a mannose-containing biotinylated albumin. The amino acid sequences of several peptides from the 35-kDa subunit showed over 85% identity to FimH, the mannose-specific adhesin of Salmonella typhimurium. Pili were labeled with 125I and examined for the ability to bind to HT-29 cells. Binding showed saturation kinetics and was inhibited by the addition of Man9(GlcNAc)2-tyrosinamide but not by oligosaccharides with fewer mannose residues. Polyclonal antibody against this 35-kDa protein also effectively blocked adhesion of pili or E. cloacae, but no effect was observed with nonspecific antibody. These studies demonstrate that the 35-kDa pilus subunit is a lectin whose specificity is directed toward Man, (GlcNAc)2 oligosaccharides.

Homonojirimycin and N-methyl-homonojirimycin inhibit N-linked oligosaccharide processing.

Homonojirimycin (HNJ) and N-methylhomonojirimycin (MHNJ) were tested as inhibitors of the purified glycoprotein processing enzymes, glucosidase I and glucosidase II. MHNJ was a reasonably good inhibitor of glucosidase I (Ki = 1 x 10(-6) M) and was about three times as effective on this enzyme as was HNJ. On the other hand, HNJ inhibited glucosidase II with a Ki of about 1 x 10(-6) M, whereas MHNJ was three times less effective (Ki = 3 x 10(-5) M). However, the butyl derivative of HNJ had very low activity toward these two processing glucosidases. HNJ and its methyl derivative were also tested in vivo using influenza virus-infected MDCK cells, and measuring the inhibition of N-linked oligosaccharide processing of the viral envelope glycoproteins. With 100 micrograms/ml of MHNJ in the medium, essentially all of the N-linked oligosaccharide chains of the virus were of the "high-mannose" type with the major structure being characterized as Glc3Man9(GlcNAc)2. Similar results were obtained with HNJ although this compound was less effective in vivo as well as in vitro. These results are in keeping with these inhibitors being effective at the glucosidase I step. Both inhibitors were also tested in MDCK cell cultures to determine whether they affected the in vivo synthesis of proteins, or of lipid-linked saccharides. In contrast to deoxynojirimycin, which has been reported to inhibit the formation of lipid-linked saccharides, no effects were seen on either the incorporation of mannose into lipid-linked saccharides or the incorporation of leucine into protein.

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.

Biological activities of the nortropane alkaloid, calystegine B2, and analogs: structure-function relationships.

Calystegines, polyhydroxy nortropane alkaloids, are a recently discovered group of plant secondary metabolites believed to influence rhizosphere ecology as nutritional sources for soil microorganisms and as glycosidase inhibitors. Evidence is presented that calystegines mediate nutritional relationships under natural conditions and that their biological activities are closely correlated with their chemical structures and stereochemistry. Assays using synthetic (+)- and (-)-enantiomers of calystegine B2 established that catabolism by Rhizobium meliloti, glycosidase inhibition, and allelopathic activities were uniquely associated with the natural, (+)-enantiomer. Furthermore, the N-methyl derivative of calystegine B2 was not catabolized by R. meliloti, and it inhibited alpha-galactosidase, but not beta-glucosidase, whereas the parent alkaloid inhibits both enzymes. This N-methyl analog therefore could serve to construct a cellular or animal model for Fabry's disease, which is caused by a lack of alpha-galactosidase activity.

Inhibition of glycoprotein processing by L-fructose and L-xylulose.

A number of unusual and rare carbohydrates were tested as potential inhibitors of various glycosidases, as well as inhibitors of N-linked oligosaccharide processing. The best inhibitors of several arylglycosidases and of glucosidase I were L-xylulose and L-fructose. Both of these sugars showed some inhibitory activity towards yeast alpha-glucosidase but were inactive against beta-glucosidase and other arylglycosidases. The inhibition of yeast alpha-glucosidase by L-xylulose was of a competitive nature and required a concentration of 1 x 10(-5) M for 50% inhibition. Both L-xylulose and L-fructose also inhibited the purified soybean glucosidase I, with 50% inhibition occurring at about 1 x 10(-4) M, but showed no inhibitory activity against soybean glucosidase II. When influenza virus-infected MDCK cells were raised in the presence of L-xylulose, there was a dose-dependent inhibition in the formation of complex types of oligosaccharides on the viral glycoproteins consistent with the inhibition of the processing glucosidase I. This inhibition resulted in the occurrence of oligosaccharides on the viral glycoproteins that were characterized as Glc3Man9(GlcNAc)2 structures. L-Fructose also inhibited glycoprotein processing in cell culture, and the inhibition resulted in the formation of similar oligosaccharides to those seen with L-xylulose. However, L-fructose was a poorer inhibitor than L-xylulose and required much higher concentrations for the same degree of inhibition. Neither of these compounds inhibited protein synthesis or the formation of lipid-linked saccharides in culture MDCK cells, even when tested at concentrations of 5 mg/ml (about 30 mM) of culture media.

Inhibition of the trehalose-P synthase of mycobacteria by various antibiotics.

A number of antibiotics were tested as potential inhibitors of the purified trehalose-P synthase of Mycobacterium smegmatis. Of about 30 compounds tested, 4 (cathomycin, circulin, diumycin, and moenomycin) were active against this enzyme. Thus each of these compounds inhibited the formation of trehalose-P by the purified trehalose-P synthase when either UDP-glucose or GDP-glucose was used as the glucosyl donor. However, preincubation of the synthase with heparin, a polyanion activator of the enzyme when UDP-glucose is used as the substrate, prevented the inhibition by these various antibiotics. Fifty percent inhibition by diumycin and moenomycin occurred at a concentration of about 50 microg/ml (Ki of about 1 x 10(-5) M), but 50% inhibition by cathomycin and circulin required substantially higher concentrations (about 50 to 200 microg/ml). The inhibition by cathomycin, diumycin, and moenomycin was of the competitive type, whereas that by circulin was noncompetitive in nature. However, the inhibition was of a complex nature and the data suggest two different binding sites for these inhibitors. Photoaffinity labeling of the synthase with an azido-UDP-[32P]glucose probe was effectively blocked by diumycin, moenomycin, or cathomycin indicating that these inhibitors do interact at the substrate binding site. These antibiotics also inhibited the growth of M. smegmatis when added to cells innoculated into trypticase soy broth. The inhibition of growth was concentration-dependent and directly proportional to the size of the bacterial innoculum. These antibiotics, however, did not inhibit protein synthesis nor did they inhibit the incorporation of mannose into lipid-linked saccharides.

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.