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 MgCl₂. 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 [³²P]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.     

Modification of Rab5 with a photoactivatable analog of geranylgeranyl diphosphate

A photoprobe analog of geranylgeranyl diphosphate (2-diazo-3,3,3-trifluoropropionyloxy-farnesyl diphosphate or DATFP-FPP) inhibits mevalonate-dependent prenylation of in vitro translated Rab5 in rabbit reticulocyte lysate, suggesting that it competes for lipid binding to the Rab geranylgeranyl transferase. Modification of Rab5 with DATFP-FPP, demonstrated by gel mobility shift and Triton X-114 phase separation experiments, confirms that the enzyme uses the analog as a substrate. The sedimentation of DATFP-modified Rab5 as a larger mass complex on sucrose density gradients indicates that it binds to other factors in rabbit reticulocyte lysate. Most importantly, DATFP-Rab5 cross-links to these soluble factors upon exposure to UV light. Immunoprecipitation with antibodies raised against proteins known to interact with Rab5 reveals that the cross-linked complexes contain Rab escort protein and GDI-1. DATFP-Rab5 also associates with membranes in a guanosine-5'-O-(3-thiotriphosphate)-stimulated manner. However, although prenylated Rab5 can be cross-linked to two unknown membrane-associated factors by the chemical cross-linker disuccinimidyl suberate, these proteins fail to be UV cross-linked to membrane-bound DATFP-Rab5. These results strongly suggest that membrane-associated factors bind Rab5 through protein-protein interactions rather than protein-prenyl interactions. The modification of Rab5 with DATFP-FPP establishes a novel photoaffinity technique for the characterization of prenyl-binding sites.     

Synthesis of a novel peptidic photoaffinity probe for the PTP-1B enzyme

The synthesis of a novel radioactive peptidic photoaffinity probe for the PTP-1B enzyme as well as some SAR leading to the choice of this compound as a photoaffinity probe are presented.     

The calcium channel blockers, 1,4-dihydropyridines, are substrates of the multidrug resistance-linked ABC drug transporter, ABCG2

The human ATP-binding cassette transporter, ABCG2, confers resistance to multiple chemotherapeutic agents and also affects the bioavailability of different drugs. [(125)I]Iodoarylazidoprazosin (IAAP) and [(3)H]azidopine were used for photoaffinity labeling of ABCG2 in this study. We show here for the first time that both of these photoaffinity analogues are transport substrates for ABCG2 and that [(3)H]azidopine can also be used to photolabel both wild-type R482-ABCG2 and mutant T482-ABCG2. We further used these assays to screen for potential substrates or modulators of ABCG2 and observed that 1,4-dihydropyridines such as nicardipine and nifedipine, which are clinically used as antihypertensive agents, inhibited the photolabeling of ABCG2 with [(125)I]IAAP and [(3)H]azidopine as well as the transport of these photoaffinity analogues by ABCG2. Furthermore, [(3)H]nitrendipine and bodipy-Fl-dihydropyridine accumulation assays showed that these compounds are transported by ABCG2. These dihydropyridines also inhibited the efflux of the known ABCG2 substrates, mitoxantrone and pheophorbide-a, from ABCG2-overexpressing cells, and nicardipine was more potent in inhibiting this transport. Both nicardipine and nifedipine stimulated the ATPase activity of ABCG2, and the nifedipine-stimulated activity was inhibited by fumitremorgin C, suggesting that these agents might interact at the same site on the transporter. In addition, nontoxic concentrations of dihydropyridines increased the sensitivity of ABCG2-expressing cells to mitoxantrone by 3-5-fold. In aggregate, results from the photoaffinity labeling and efflux assays using [(125)I]IAAP and [(3)H]azidopine demonstrate that 1,4-dihydropyridines are substrates of ABCG2 and that these photolabels can be used to screen new substrates and/or inhibitors of this transporter.     

Application of 5-azido-UDP-glucose and 5-azido-UDP-glucuronic acid photoaffinity probes for the determination of the active site orientation of microsomal UDP-glucosyltransferases and UDP-glucuronosyltransferases.

A new approach to determining the active site orientation of microsomal glycosyltransferases is presented which utilizes the photoaffinity analogs [32P]5-Azido-UDP-glucose ([32P]5N3UDP-Glc) and [32P]5-Azido-UDP-glucuronic acid ([32P]5N3UDP-GlcA). It was previously shown that both photoprobes could be used to photolabel UDP-glucose:dolichol phosphate glucosyltransferase (Glc-P-Dol synthase), as well as the family of UDP-glucuronosyltransferases in rat liver microsomes. The effects of detergents, proteases, and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) on the photolabeling of these enzymes were examined in intact rat liver microsomes. Photolabeling of Glc-P-Dol synthase by either photoprobe was the same in intact or disrupted vesicles, was susceptible to trypsin digestion, and was inhibited by the nonpenetrating inhibitor DIDS. Photolabeling of the UDP-glucuronosyltransferases by [32P]5N3UDP-GlcA was stimulated 1.3-fold in disrupted vesicles as compared to intact vesicles, whereas photolabeling of these enzymes by [32P]5N3UDP-Glc showed a 14-fold increase when vesicles were disrupted. Photolabeled UDP-glucuronosyltransferases were only susceptible to trypsin digestion in disrupted vesicles, and this was further verified by Western blot analyses. The results indicate a cytoplasmic orientation for access of UDP-sugars to Glc-P-Dol synthase and a lumenal orientation of most UDP-glucuronosyltransferases.     

Synthesis and biological evaluation of didemnin photoaffinity analogues.

The synthesis of four benzophenone-containing analogues of the antiproliferative natural product didemnin B is presented. In vitro protein biosynthesis inhibition potency and antitumor activity were evaluated. The results indicate that all four analogues are biologically active and could serve as photoaffinity reagents for the study of receptor-binding interactions of didemnins. These analogues could also be useful in studying antitumor effects of didemnins.     

Variability of human hepatic UDP-glucuronosyltransferase activity.

The availability of a unique series of liver samples from human subjects, both control patients (9) and those with liver disease (6; biliary atresia (2), retransplant, chronic tyrosinemia type I, tyrosinemia, Wilson's disease) allowed us to characterize human hepatic UDP-glucuronosyltransferases using photoaffinity labeling, immunoblotting and enzymatic assays. There was wide inter-individual variation in photoincorporation of the photoaffinity analogs, [32P]5-azido-UDP-glucuronic acid and [32P]5-azido-UDP-glucose and enzymatic glucuronidation of substrates specific to the two subfamilies of UDP-glucuronosyltransferases. However, the largest differences were between subjects with liver disease. Glucuronidation activities toward one substrate from each of the UDP-glucuronosyltransferases subfamilies, 1A and 2B, for control and liver disease, respectively, were 1.7-4.5 vs 0.4-4.7 nmol/mg x min for hyodeoxycholic acid (2B substrate) and 9.2-27.9 vs 8.1-75 nmol/mg x min for pchloro-m-xylenol (1A substrate). Microsomes from a patient with chronic tyrosinemia (HL32) photoincorporated [32P]5-azido-UDP-glucuronic acid at a level 1.5 times higher than the other samples, was intensely photolabeled by [32P]5-azido-UDP-glucose and had significantly higher enzymatic activity toward p-chloro-m-xylenol. Immunoblot analysis using anti-UDP-glucuronosyltransferase antibodies demonstrated wide inter-individual variations in UDP-glucuronosyltransferase protein with increased UDP-glucuronosyltransferase protein in HL32 microsomes, corresponding to one of the bands photolabeled by both probes. Detailed investigation of substrate specificity, using substrates representative of both the 1A (bilirubin, 4-nitrophenol) and 2B (androsterone, testosterone) families was carried out with HL32, HL38 (age and sex matched control) and HL18 (older control). Strikingly increased (5-8-fold) glucuronidation activity was seen in comparison to HL18 only with the phenolic substrates. The results indicate that one or more phenol-specific UDP-glucuronosyltransferase 1A isoforms are expressed at above normal levels in this tyrosinemic subject.     

3'-O-(4-benzoyl)benzoylcytidine 5'-triphosphate. A substrate and photoaffinity label for CMP-N-acetylneuraminic acid synthetase

A photoreactive, radiolabeled pyrimidine nucleotide, 3'-O-(4-benzoyl)benzoylcytidine 5'-triphosphate was synthesized from benzoylbenzoic acid and radiolabeled CTP. Benzoylbenzoyl-[5-3H]CTP could substitute for CTP, in an enzymatic reaction with N-acetylneuraminic acid catalyzed by Escherichia coli or rat liver CMP-NeuAc synthetase, to yield radiolabeled benzoyl-benzoyl-CMP-NeuAc. E. coli CMP-NeuAc synthetase could be specifically radiolabeled using benzoylbenzoyl-[alpha-32P]CTP as a photoaffinity label. This specific covalent binding occurred using enzyme preparations of different degrees of purity. These results suggest that benzoylbenzoic acid derivatives of pyrimidines should be of general use in the identification and active site mapping of pyrimidine-requiring proteins and enzymes. These include glycosyltransferases, sugar nucleotide synthetases, and transporters, and enzymes participating in the conjugation of bile acids and biosynthesis of nucleic acids and choline nucleotides.     

Disaccharide analogs as probes for glycosyltransferases in Mycobacterium tuberculosis

Glycosyltransferases (GTs) play a crucial role in mycobacterial cell wall biosynthesis and are necessary for the survival of mycobacteria. Hence, these enzymes are potential new drug targets for the treatment of tuberculosis (TB), especially multiple drug-resistant TB (MDR-TB). Herein, we report the efficient syntheses of Araf(alpha 1-->5)Araf, Galf(beta 1-->5)Galf, and Galf(beta 1-->6)Galf disaccharides possessing a 5-N,N-dimethylaminonaphthalene-1-sulfonamidoethyl (dansyl) unit that were prepared as fluorescent disaccharide acceptors for arabinosyl- and galactosyl-transferases, respectively. Such analogs may offer advantages relative to radiolabeled acceptors or donors for studying the enzymes and for assay development and compound screening. Additionally, analogs possessing a 5-azidonaphthalene-1-sulfonamidoethyl unit were prepared as photoaffinity probes for their potential utility in studying active site labeling of the GTs (arabinosyl and galactosyl) in Mycobacterium tuberculosis (MTB). Beyond their preparation, initial biological testing and kinetic analysis of these disaccharides as acceptors toward glycosyltransferases are also presented.     

Advances in flavonoid glycosyltransferase research: integrating recent findings with long-term citrus studies

Flavonoid glycosides are required for a number of crucial roles in planta and have the potential for development in a variety of agricultural, medicinal, and biotechnological applications. A number of recent advancements have been made in characterizing glycosyltransferases, the enzymes that are responsible for the synthesis of these important molecules. In this review, glycosyltransferases are considered with regard to biochemical properties, expression patterns, levels of enzyme activity during development, and structure/function relationships. This is presented with historical context to highlight critical findings, particularly with regard to the innovative work that has come from research on citrus species. The plant glycosyltransferase crystal structures that have been solved over the past decade, either alone or in complex with sugar donor and/or acceptor molecules, are discussed. The application of results from these structures to inform current structure/function work as well as implications and goals for future crystallography and tertiary modeling studies are considered. A thorough understanding of the properties of glycosyltransferases will be a critical step in any future biotechnological application of these enzymes in areas such as crop improvement and custom design of enzymes to produce desired compounds for nutritional and/or medicinal usage.     

Tautomerism of 2-azidoadenine nucleotides. Effects on enzyme kinetics and photoaffinity labeling

The 2-azidoadenine nucleotides show promise as photoaffinity probes. Substitution at the C-2 position should favor an anti conformation and enable binding of the analogue to enzyme sites which exhibit low affinity for the 8-azidoadenine derivatives. The 2-azidoadenine nucleotides were found to be substrates for pyruvate kinase, phosphofructokinase, adenylate kinase, hexokinase and the mitochondrial F1-ATPase. However, tautomerism of 2-azidoadenine nucleotides to two nonphotoreactive tetrazole forms complicates kinetic analyses and their use as photoaffinity probes. An analysis of the ultraviolet spectra of these analogues enables an estimation of the tetrazolo isomer content and the rates of tautomerization. The photoreactive azido isomer was found to represent only 45% of the total analogue population in neutral aqueous solution. The azidoazomethine-tetrazole equilibrium favors the azido isomer in acidic or nonpolar solutions. The first-order rate constants at 25 degrees C were determined to be 0.017 min-1 and 0.021 min-1 for tautomerism to the azido and tetrazolo isomers, respectively. Prior equilibration of the probe in various solvents thus allows investigation of the analogue's behavior with an enzyme system at different, essentially fixed, isomer ratios. The determination of the impact of the tetrazolo tautomers on the system allows optimization of conditions for photoaffinity-labeling experiments.     

Structure and mechanism of GumK, a membrane-associated glucuronosyltransferase

Xanthomonas campestris GumK (beta-1,2-glucuronosyltransferase) is a 44-kDa membrane-associated protein that is involved in the biosynthesis of xanthan, an exopolysaccharide crucial for this bacterium's phytopathogenicity. Xanthan also has many important industrial applications. The GumK enzyme is the founding member of the glycosyltransferase family 70 of carbohydrate-active enzymes, which is composed of bacterial glycosyltransferases involved in exopolysaccharide synthesis. No x-ray structures have been reported for this family. To better understand the mechanism of action of the bacterial glycosyltransferases in this family, the x-ray crystal structure of apo-GumK was solved at 1.9A resolution. The enzyme has two well defined Rossmann domains with a catalytic cleft between them, which is a typical feature of the glycosyltransferase B superfamily. Additionally, the crystal structure of GumK complexed with UDP was solved at 2.28A resolution. We identified a number of catalytically important residues, including Asp(157), which serves as the general base in the transfer reaction. Residues Met(231), Met(273), Glu(272), Tyr(292), Met(306), Lys(307), and Gln(310) interact with UDP, and mutation of these residues affected protein activity both in vitro and in vivo. The biological and structural data reported here shed light on the molecular basis for donor and acceptor selectivity in this glycosyltransferase family. These results also provide a rationale to obtain new polysaccharides by varying residues in the conserved alpha/beta/alpha structural motif of GumK.     

Identification and solubilization of a cAMP-independent protein kinase from mouse L-cell smooth membranes

1. Smooth membranes have been prepared from mouse L-cells and found to contain an endogenous protein kinase activity. 2. The enzyme(s) responsible for this activity use ATP, but no other nucleoside triphosphates, to phosphorylate endogenous membrane proteins as well as exogenously-added protein substrates such as phosvitin and casein. 3. Mg2+ is required for enzyme activity, maximal activity is observed at pH 7.5-8.0 and the kinase is not dependent on, or stimulated by, cyclic 3'-5' AMP. 4. The kinase activity is not decreased by the Walsh heat-stable inhibitor of cyclic 3'-5' AMP-dependent protein kinases. 5. Fifty percent or more of the membrane-associated kinase activity can be solubilized by extracting membranes with buffer containing 0.6 M NaCl. 6. The solubilized enzyme resembles the membrane-associated activity in its Mg2+ requirement, pH optimum and independence of cyclic 3'-5' AMP. 7. Phosvitin and casein are better exogenous substrates than histones or protamine for phosphorylation by the enzyme in either the membrane-associated or solubilized state.     

Azido derivative of tricarboxylic acid for photoaffinity labeling

A new photoaffinity probe, 5-(1-hydroxy-4-azidophenylazo)-1,2,3-benzenetricarboxylic acid, was synthesized and characterized. This reagent can be potentially used in photoaffinity labeling of the mitochondrial tricarboxylate carrier, as well as of enzymes interacting with tricarboxylic acids. Inhibition and labeling of the mitochondrial tricarboxylate carrier is presented.     

Novel ligands for the affinity labelling of luteinizing hormone releasing hormone receptors.

A number of novel luteinizing hormone releasing hormone (LHRH) analogues incorporating biotin together with potential covalent attachment sites have been synthesized. Those based on the des-Gly10-[D-Lys6]-LHRH ethylamide peptide backbone resulted in the most useful characteristics of binding to the LHRH receptor in rat anterior pituitary gland membranes. Of these, des-Gly10-[biotinyl-aminoethylglycyl-D-Lys6]-LHRH ethylamide (XBAL) gave the best specific: non-specific binding ratio, with 44 +/- 6% (+/- S.E.M.) of total binding being specific with a Kd of 131 +/- 16 pM (+/- S.E.M., n = 4) as determined by Scatchard analysis. Two methods have been used to covalently crosslink these analogues with the LHRH receptor; photoaffinity labelling and the use of homobifunctional N-hydroxysuccinimide ester crosslinkers. The photoaffinity analogues gave poor specific: non-specific binding ratios. Of the chemical crosslinkers tested, ethylene glycolbis(succinimidylsuccinate) (EGS) was found to be the most efficient at covalently linking the 125I-XBAL bound to the LHRH receptor site. At an EGS concentration of 5 mM, 23 +/- 3% (+/- S.E.M.) of the specific binding of 125I-XBAL was covalently crosslinked.     

Synthesis and enzymatic evaluation of modified acceptors of recombinant blood group A and B glycosyltransferases

The disaccharide alpha-L-Fucp-(1 --> 2)-beta-D-Galp-(1 --> O)-Octyl (1) is an acceptor for the human blood group A and B glycosyltransferases. Seven analogues of 1, containing deoxy, methoxy and arabino modifications of the Fuc residue, were chemically synthesized and kinetically evaluated in radioactive enzymatic assays. Both the enzymes tolerate modification of the 3'-OH on the fucose residue. The 2'-OH was found to be key to the recognition of the acceptors by these enzymes. The arabino derivative was recognized as an acceptor by the A transferase (Km of 200 microM), but not the B transferase and is the first synthetic acceptor capable of distinguishing between the two enzyme activities.     

Bicelles in structure-function studies of membrane-associated proteins

Bicelles are a novel form of long-chain/short-chain phospholipid aggregates, which are useful for biophysical and biochemical studies of membrane-associated biomolecules. In this work, we review the development of bicelles and their uses in structural characterization (primarily via NMR, circular dichroism, and fluorescence) of membrane-associated peptides. We also show that bicellar phospholipids are substrates for lipolytic enzymes. For this latter work, we employed a 31P NMR enzymatic assay system to examine the kinetic behavior of cobra venom phospholipase A(2) toward a variety of bicellar substrates. This enzyme hydrolyzed all bicelle lipids at rates comparable to those found for the enzyme action on traditional micellar substrates, which are the best substrates for this enzyme. In addition, we found that this PLA(2) showed no significant preference for long-chain or short-chain phospholipids when they were presented as mixtures in bicelles.     

Tautomerism of 2-azidoadenine nucleotides Effects on enzyme kinetics and photoaffinity labeling

The 2-azidoadenine nucleotides show promise as photoaffinity probes. Substitution at the C-2 position should favor an anti conformation and enable binding of the analogue to enzyme sites which exhibit low affinity for the 8-azidoadenine derivatives. The 2-azidoadenine nucleotides were found to be substrates for pyruvate kinase, phosphofructokinase, adenylate kinase, hexokinase and the mitochondrial F₁-ATPase. However, tautomerism of 2-azidoadenine nucleotides to two nonphotoreactive tetrazole forms complicates kinetic analyses and their use as photoaffinity probes. An analysis of the ultraviolet spectra of these analogues enables an estimation of the tetrazolo isomer content and the rates of tautomerization. The photoreactive azido isomer was found to represent only 45% of the total analogue population in neutral aqueous solution. The azidoazomethine-tetrazole equilibrium favors the azido isomer in acidic or nonpolar solutions. The first-order rate constants at 25°C were determined to be 0.017 min^?1 and 0.021 min^?1 for tautomerism to the azido and tetrazolo isomers, respectively. Prior equilibration of the probe in various solvents thus allows investigation of the analogue's behavior with an enzyme system at different, essentially fixed, isomer ratios. The determination of the impact of the tetrazolo tautomers on the system allows optimization of conditions for photoaffinity-labeling experiments.