Purification and characterization of the bacterial UDP-GlcNAc:undecaprenyl-phosphate GlcNAc-1-phosphate transferase WecA

To date, the structural and functional characterization of proteins belonging to the polyprenyl-phosphate N-acetylhexosamine-1-phosphate transferase superfamily has been relentlessly held back by problems encountered with their overexpression and purification. In the present work and for the first time, the integral membrane protein WecA that catalyzes the transfer of the GlcNAc-1-phosphate moiety from UDP-GlcNAc onto the carrier lipid undecaprenyl phosphate, yielding undecaprenyl-pyrophosphoryl-GlcNAc, the lipid intermediate involved in the synthesis of various bacterial cell envelope components, was overproduced and purified to near homogeneity in milligram quantities. An enzymatic assay was developed, and the kinetic parameters of WecA as well as the effects of pH, salts, cations, detergents, and temperature on the enzyme activity were determined. A minimal length of 35 carbons was required for the lipid substrate, and tunicamycin was shown to inhibit the enzyme at submicromolar concentrations.     

Specific lipids enhance the activity of UDP-GlcNAc: dolichol phosphate GlcNAc-1-phosphate transferase in rat liver endoplasmic reticulum membrane vesicles.

The rate of the reaction catalyzed by UDP-N-acetylglucosamine (GlcNAc):dolichol phosphate GlcNAc-1-phosphate transferase in rat liver endoplasmic reticulum vesicles was shown to be influenced by particular lipids. Utilizing in vitro assay conditions where the membrane vesicles retained latency of glucose-6-phosphatase activity, the addition of phosphatidylethanolamine, cardiolipin, or monogalactosyldiglyceride resulted in severalfold increases in the rate of dolichol pyrophosphate N-acetylglucosamine synthesis. Other phospholipids were not stimulatory. These rates were dependent on the concentrations of the exogenous lipids and of the substrate dolichol phosphate. In the presence of cardiolipin, the membrane-bound enzyme became more susceptible to inactivation by protease K and to inhibition by tunicamycin. Titration of cardiolipin-containing endoplasmic reticulum vesicles with adriamycin indicated that the majority of the cardiolipin was exposed on the outer surface. These results suggest that the particular lipids altered membrane structure in a way that allowed further access of the enzyme to substrate, inhibitor, and other molecules. Lipids observed in these studies to be stimulatory are known to exist in the macromolecular hexagonal phase and may therefore be affecting the GlcNAc-1-phosphate transferase by locally disrupting the bilayer structure of the membrane. As other dolichol-utilizing enzymes have been previously observed by other investigators to be similarly influenced by such lipids, the effects may be common to enzymes of the dolichol cycle.     

Conserved sequences in enzymes of the UDP-GlcNAc/MurNAc family are essential in hamster UDP-GlcNAc:dolichol-P GlcNAc-1-P transferase

The UDP-GlcNAc/MurNAc family of eukaryotic and prokaryotic enzymes use UDP-GlcNAc or UDP-MurNAc-pentapeptide as donors, dolichol-P or polyprenol-P as acceptors, and generate sugar-P-P-polyisoprenols. A series of six conserved sequences, designated A through F and ranging from 5 to 13 amino acid residues, has been identified in this family. To determine whether these conserved sequences are required for enzyme function, various mutations were examined in hamster UDP- GlcNAc:dolichol-P GlcNAc-1-P transferase (GPT). Scramble mutations of sequences B-F, generated by scrambling the residues within each sequence, demonstrated that each is important in GPT. While E and F scrambles appeared to prevent stable expression of GPT, scrambling of B- D resulted in GPT mutants that could be stably expressed and bound tunicamycin, but lacked enzymatic activity. Further, the C and D scramble mutants had an unexpected sorting defect. Replacement of sequences B-F with prokaryotic counterparts from either the B.subtilis mraY or E.coli rfe genes also affected GPT by preventing expression of the mutant protein (B, F) or inhibiting its enzymatic activity (C-E). For the C-E replacements, no acquisition of acceptor activity for polyprenol-P, the fully unsaturated natural bacterial acceptor, was detected. These studies show that the conserved sequences of the UDP- GlcNAc/MurNAc family are important, and that the eukaryotic and prokaryotic counterparts are not freely interchangeable. Since several mutants were efficiently expressed and bound tunicamycin, yet lacked enzymatic activity, the data are consistent with these sequences having a direct role in product formation.      

Evidence that the hamster tunicamycin resistance gene encodes UDP-GlcNAc:dolichol phosphate N-acetylglucosamine-1-phosphate transferase.

A cDNA clone isolated from Chinese hamster ovary cells conferred elevated GlcNAc-1-P-transferase (GPT) activity and resistance to tunicamycin in transfected cells (Zhu, X., and Lehrman, M. A. (1990) J. Biol. Chem. 265, 14250-14255). It had been assumed that this cDNA, termed TRG for tunicamycin resistance gene, encoded GPT enzyme. However, other functions were not ruled out. Thus, by one of several mechanisms, the TRG protein could have instead functioned by activation of the transfected host's endogenous GPT enzyme. To analyze the biochemical function of the TRG protein, hamster TRG cDNA was stably expressed at high levels in Chinese hamster ovary cells. In addition, several antipeptide polyclonal antibodies directed against the predicted TRG protein were obtained. With these tools in hand, experiments were performed to test the hypothesis that the TRG encodes GPT enzyme, as well as to rule out other possible functions for the TRG protein. These experiments included examination of the effects of solubilization of membranes on TRG-dependent GPT activity, the apparent binding of tunicamycin to the TRG protein, and the immunoadsorption of GPT activity with TRG protein-specific antibodies. From these results, we conclude that the hamster TRG most likely encodes GPT enzyme.     

D-glyceraldehyde-3-phosphate dehydrogenase from HeLa cells--1. Purification and properties of the enzyme.

1. D-GPDH from HeLa cells was isolated and purified. 2. Some basic kinetic constants are reported. 3. Sodium dodecyl polyacrylamide gel electrophoresis gave a single band with a molecular weight of approximately 36 K. 4. ATP and NADH inhibit competitively enzyme activity. 5. Comparative catalytic properties of GPDH from normal and tumor cells were effectuated.     

Purification, properties, and isozyme pattern of galactose-1-phosphate uridyl transferase from calf liver.

Galactose-1-phosphate uridyl transferase was purified approximately 2000-fold from calf liver with a yield of 15%. The purification procedure involved ammonium sulfate fractionation, calcium phosphate-gel adsorption, and chromatography on DEAE-cellulose, hydroxylapatite, and Sephadex columns. The purified product demonstrated five protein bands on polyacrylamide-gel electrophoresis. Each band had transferase activity as five peaks of activity were observed on preparative polyacrylamide-gel electrophoresis. Galactose-1-phosphate uridyl transferase showed no requirement for divalent metals for activity. In contrast, it was inhibited by Mg²⁺ and other divalent metals. The purified enzyme but not the crude preparation was stimulated by sulfhydryl compounds. The enzyme was completely inhibited by low concentrations of p-hydroxymercuribenzoate.     

Muscle D-glyceraldehyde-3-phosphate dehydrogenase from Anas sp.--1. Purification and properties of the enzyme

Muscle D-GPDH from Anas sp. ws isolated and purified. Basic kinetic constants and physico-chemical studies are reported. The enzyme is a tetramer with a mol. wt of 140,000. Sodium dodecyl sulfate polyacrylamide gel electrophoresis gave s single band with a mol. wt of approx. 36,000. The other catalytic properties of the enzyme are discussed relative to those of enzyme obtained from other sources.     

Cloning, sequence, and expression of a cDNA encoding hamster UDP-GlcNAc:dolichol phosphate N-acetylglucosamine-1-phosphate transferase

UDP-GlcNAc:dolichol phosphate N-acetylglucosamine-1-phosphate transferase (GPT) catalyzes the initial reaction required for synthesis of dolichol-P-P-oligosaccharides. We report here on the sequence and expression of a full-length cDNA clone encoding hamster GPT. The cDNA predicts a protein of 408 amino acid residues including 10 hydrophobic segments. Several portions of the hamster GPT sequence constituting one-third of the protein have 60% or greater identity with yeast GPT, and one-half of the conserved sequence falls within the hydrophobic segments. In addition, hamster GPT has two copies of a putative dolichol recognition sequence recently identified in three yeast enzymes that interact with dolichol. The protein lacks KDEL or DEKKMP-type carboxyl-terminal ER sorting sequences. When expressed in COS-1 cells, the cDNA causes a 5-7-fold increase of GPT activity in membrane fractions. The activity was completely inhibitable by tunicamycin, and the primary product was shown to be GlcNAc-pyrophosphoryldolichol. This cDNA represents the first enzyme of the dolichol-oligosaccharide biosynthetic pathway to be cloned from a vertebrate source and demonstrates structural homology between the enzymes of the yeast and mammalian pathways.     

Coupling of the dolichol-P-P-oligosaccharide pathway to translation by perturbation-sensitive regulation of the initiating enzyme,GlcNAc-1-P transferase

In mammalian cells, inhibition of translation interferes with synthesis of the lipid-linked oligosaccharide (LLO) Glc3Man9GlcNAc2-P-P-dolichol as measured with radioactive sugar precursors. Conflicting hypotheses have been proposed, and the fundamental basis for this regulation has remained elusive. Here, fluorophore-assisted carbohydrate electrophoresis (FACE) was used to measure LLO concentrations directly in cells treated with translation blockers. Further, LLO biosynthetic enzymes were assayed in vitro with endogenous acceptor substrates using either cells gently permeabilized with streptolysin-O (SLO) or microsomes from homogenized cells. In Chinese hamster ovary (CHO)-K1 cells treated with translation blockers, FACE did not detect changes in concentrations of Glc3Man9GlcNAc2-P-P-dolichol or early LLO intermediates. These results do not support earlier proposals for feedback repression of LLO initiation by accumulated Glc3Man9GlcNAc2-P-P-dolichol, or inhibition of a GDP-mannose dependent transferase. With microsomes from cells treated with translation blockers, there was no interference with LLO initiation by GlcNAc-1-P transferase (GPT), mannose-P-dolichol synthase, glucose-P-dolichol synthase, or LLO synthesis in vitro, as reported previously. Surprisingly, inhibition of all of these was detected with the SLO in vitro system. Additional experiments with the SLO system showed that the three transferases shared a limited pool of dolichol-P that was trapped as Glc3Man9GlcNAc2-P-P-dolichol by translation arrest. Overexpression of GPT was unable to reverse the effects of translation arrest on LLO initiation, and experiments with FACE and the SLO system showed that overexpressed GPT was not functional in vivo, although it was highly active in microsomal assays. Thus, the combined use of the SLO in vitro system and FACE showed that LLO biosynthesis depends upon a limited primary pool of dolichol-P. Physical perturbation associated with microsome preparation appears to make available a secondary pool of dolichol-P, masking inhibition by translation arrest, as well as activating a nonfunctional fraction of GPT. The implications of these results for the organization of the LLO pathway are discussed.     

Identification of the gonococcal glmU gene encoding the enzyme N-acetylglucosamine 1-phosphate uridyltransferase involved in the synthesis of UDP-GlcNAc.

In searching for the gonococcal sialyltransferase gene(s), we cloned a 3.8-kb DNA fragment from gonococcus strain MS11 that hybridized with the oligonucleotide JU07, which was derived from the conserved C terminus of the sialyl motif present in mammalian sialyltransferases. Sequencing of the fragment revealed four putative open reading frames (ORFs), one of which (ORF-1) contained a partial sialyl motif including the amino acid sequence VGSKT, which is highly conserved among sialyltransferases. The gene was flanked by two inverted repeats containing the neisserial DNA uptake sequence and was preceded by a putative sigma 54 promoter. Database searches, however, revealed a high degree of homology between ORF-1 and the N-acetylglucosamine 1-phosphate uridyltransferase (GlmU) of Escherichia coli and Bacillus subtilis and not with any known sialyltransferase. This homology was further established by the successful complementation of an orf-1 mutation by the E. coli glmU gene. Enzyme assays demonstrated that ORF-1 did not possess sialyltransferase activity but mimicked GlmU function catalyzing the conversion of N-acetylglucosamine 1-phosphate into UDP-N-acetylglucosamine, which is a key metabolite in the syntheses of lipopolysaccharide, peptidoglycan, and sialic acids.     

Activation and inhibition of microsomal glutathione transferase from mouse liver.

Mouse liver microsomal glutathione transferase was purified in an N-ethylmaleimide-activated as well as an unactivated form. The enzyme had a molecular mass of 17 kDa and a pI of 8.8. It showed cross-reactivity with antibodies raised against rat liver microsomal glutathione transferase, but not with any of the available antisera raised against cytosolic glutathione transferases. The fully N-ethylmaleimide-activated enzyme could be further activated 1.5-fold by inclusion of 1 microM-bromosulphophthalein in the assay system. The latter effect was reversible, which was not the case for the N-ethylmaleimide activation. At 20 microM-bromosulphophthalein the activated microsomal glutathione transferase was strongly inhibited, while the unactivated form was activated 2.5-fold. Inhibitors of the microsomal glutathione transferase from mouse liver showed either about the same I50 values for the activated and the unactivated form of the enzyme, or significantly lower I50 values for the activated form compared with the unactivated form. The low I50 values and the steep slope of the activity-versus-inhibitor-concentration curves for the latter group of inhibitors tested on the activated enzyme indicate a co-operative effect involving conversion of activated enzyme into the unactivated form, as well as conventional inhibition of the enzyme.     

CDP-diglyceride:inositol transferase from rat liver. Purification and properties.

CDP-diglyceride:inositol transferase, which catalyzes the final step of the de novo synthesis of phosphatidylinositol, was solubilized by sodium cholate from microsomes prepared from rat liver and purified by ammonium sulfate fractionation, sucrose density gradient centrifugation, and DEAE-cellulose column chromatography. Addition of phospholipid during the purification and the assay procedures prevented irreversible loss of the enzyme activity to some extent. The resulting preparation was nearly homogeneous as judged by polyacrylamide gel electrophoresis. The recovery of the purified enzyme from the microsomal fraction was 3 to 3.3% with respect to activity and 0.12% with respect to amount of protein. The molecular weight of the enzyme was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 60,000. The purified enzyme required exogenous phospholipds for its activity. Various phospholipid classes activated the enzyme rather nonspecifically. The Km for myo-inositol was 2.5 X 10(-3) M and that for CDP-diglyceride was 1.7 X 10(-4) M. The pH optimum was 8.6. The enzyme required Mm2+ or Mg2+ for activity. The optimal concentration of Mn2+ for activation was 0.5 mM, while the activity in the presence of Mg2+ increased up to 20 mM. The enzyme was inhibited by thiol-reactive reagents. There was a competition for inositol by inosose-2 but not by scyllitol.     

Purification and properties of D-mannitol-1-phosphate dehydrogenase and D-glucitol-6-phosphate dehydrogenase from Escherichia coli.

D-Mannitol-1-phosphate dehydrogenase (EC 1.1.1.17) and D-glucitol-6-phosphate dehydrogenase (EC 1.1.1.140) were purified to apparent homogeneity in good yields from Escherichia coli. The amino acid compositions, N-terminal amino acid sequences, sensitivities to chemical reagents, and catalytic properties of the two enzymes were determined. Both enzymes showed absolute specificities for their substrates. The subunit molecular weights of mannitol-1-phosphate and glucitol-6-phosphate dehydrogenases were 40,000 and 26,000, respectively; the apparent molecular weights of the native proteins, determined by gel filtration, were 40,000 and 117,000, respectively. It is therefore concluded that whereas mannitol-1-phosphate dehydrogenase is a monomer, glucitol-6-phosphate dehydrogenase is probably a tetramer. These two proteins differed in several fundamental respects.     

Human cathepsin B1. Purification and some properties of the enzyme

1. Cathepsin B1 was purified from human liver by a method involving autolysis, fractional precipitation with acetone, adsorption on, and stepwise elution from, CM-cellulose and an organomercurial adsorbent, gel chromatography and finally equilibrium chromatography on CM-cellulose. 2. The early stages of the procedure, including the use of the organomercurial adsorbent, were suitable for the simultaneous isolation of cathepsin D. The two cathepsins were sharply separated on the organomercurial column, and particular attention was given to the method for the preparation and use of this adsorbent. 3. A method is described for the staining of analytical isoelectric-focusing gels for cathepsin B1 activity, as well as protein. By this method it was shown that cathepsin B1 was represented by at least six isoenzymes during the greater part of the purification procedure. After the gel-chromatography step this group of isoenzymes was obtained essentially free of other proteins, in good yield. The isoenzymes were resolved from this mixture by chromatography on CM-cellulose. The purified enzyme was stable for several weeks at slightly acid pH values in the absence of thiol compounds; it was unstable above pH7. 4. The pI values of the isoenzymes of cathepsin B1 extended from pH4.5 to 5.5, that of the major isoenzyme tending to increase from 5.0 to 5.2 during the purification procedure. Gel chromatography indicated a molecular weight of 27500 for all of the isoenzymes, whereas polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate gave a value of 24000. 5. An antiserum raised in sheep against the purified enzyme reacted specifically with the alkali-denatured molecule. Purified cathepsin B1 contained no material precipitable by an anti-(human cathepsin D) serum. 6. The enzyme hydrolysed several N-substituted derivatives of l-arginine 2-naphthylamide, as well as haemoglobin, azo-haemoglobin, azo-globin and azo-casein. Greatest activity was obtained near pH6.0. 7. The sensitivity of human cathepsin B1 to chemical inhibitors was generally similar to that of other thiol proteinases. The enzyme was inactivated by the chloromethyl ketones derived from tosylphenylalanine, tosyl-lysine, acetyltetra-alanine and acetyldialanylprolylalanine. 8. The hydrolysis of alpha-N-benzoyl-dl-arginine 2-naphthylamide by extracts of human liver at pH6 was attributable entirely to cathepsin B1.     

5-Enolpyruvylshikimate-3-phosphate synthase of Klebsiella pneumoniae. 1. Purification and properties

The shikimate pathway enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase (3-phosphoshikimate 1-carboxyvinyltransferase, EC 2.5.1.19) has been purified to apparent homogeneity from Aerobacter aerogenes, strain 62-1 (= Klebsiella pneumoniae ATCC 25306). A 3300-fold purification of the enzyme was achieved by ammonium sulfate fractionation, heat precipitation, chromatography on DEAE-cellulose, Sephadex G-75, and cellulose phosphate, and chromatofocusing as the final step. The recovery was 49%. An apparent relative molecular mass of 32400 was determined by calibrated gel filtration, while a single peptide chain of Mr = 42900 was found by sodium dodecyl sulfate/acrylamide gel electrophoresis. The isoelectric point was determined to be at pH 4.6. Two distinct pH optima (pH 5.4 and 6.8) were observed for the enzyme-catalyzed formation of EPSP from phosphoenolpyruvate (PEP) and shikimate 3-phosphate(S3P). For the reverse reaction, the pH optima were 5.6 and 7.6. No evidence for a metal cofactor was found. While the temperature optimum was at 60 degrees C, the activation energies were calculated to be 54.2 kJ/mol for the forward, and 64.1 kJ/mol for the reverse reaction. At low PEP and S3P concentrations, anions acted as activators of EPSP synthase at low concentrations, and as inhibitors at high concentrations. Non-linear Lineweaver-Burk plots were interpreted to result from the activation of EPSP synthase by its anionic substrates. The following dissociation constants were determined for the respective enzyme-substrate complexes: forward reaction: 43 microM (PEP) and 22 microM (S3P); reverse reaction: 1.3 microM (EPSP) and 2.6 mM (Pi). The kinetic patterns indicate a random sequential mechanism for the forward reaction.