Novel amide-based inhibitors of inosine 5'-monophosphate dehydrogenase

A series of novel amide-based small molecule inhibitors of inosine monophosphate dehydrogenase (IMPDH) was explored. The synthesis and the structure-activity relationships (SARs) derived from in vitro studies are described.     

Kinetic mechanism of Tritrichomonas foetus inosine 5'-monophosphate dehydrogenase

IMP dehydrogenase (IMPDH) catalyzes the oxidation of IMP to XMP with conversion of NAD+ to NADH. This reaction is the rate-limiting step in de novo guanine nucleotide biosynthesis. IMPDH is a target for antitumor, antiviral, and immunosuppressive chemotherapy. We have determined the complete kinetic mechanism for IMPDH from Tritrichomonas foetus using ligand binding, isotope effect, pre-steady-state kinetic, and rapid quench kinetic experiments. Both substrates bind to the free enzyme, which suggests a random mechanism. IMP binds to the enzyme in two steps. Two steps are also involved when IMP binds to a mutant IMPDH in which the active site Cys is substituted with a Ser. This observation suggests that this second step may be a conformational change of the enzyme. No Vm isotope effect is observed when [2-2H]IMP is the substrate which indicates that hydride transfer is not rate-limiting. This result is confirmed by the observation of a pre-steady-state burst of NADH production when monitored by absorbance. However, when NADH production was monitored by fluorescence, the rate constant for the exponential phase is 5-10-fold lower than when measured by absorbance. This observation suggests that the fluorescence of enzyme-bound NADH is quenched and that this transient represents NADH release from the enzyme. The time-dependent formation and decay of [14C]E-XMP intermediates was monitored using rapid quench kinetics. These experiments indicate that both NADH release and E-XMP hydrolysis are rate-limiting and suggest that NADH release precedes hydrolysis of E-XMP.     

Direct assay method for inosine 5'-monophosphate dehydrogenase activity

A rapid microassay method for the accurate measurement of the activity of inosine 5'-monophosphate dehydrogenase in crude tissue extracts was described. [8-14C]IMP and the radioactive products were separated by high-voltage electrophoresis in 0.1 M potassium phosphate buffer, pH 7.0, for 45 min. This separation method provides an analysis of the possible interfering reactions such as the metabolic conversion of the substrate IMP to inosine and adenylosuccinate, and the loss of the product XMP to xanthosine or GMP and to other metabolites. Low blank values were consistently obtained with this method because the XMP spot moves faster than the IMP spot. The major advantages of this assay method are direct measurement of IMP dehydrogenase activity in crude extracts, high sensitivity (with a limit of detection of 5 pmol of XMP production), high reproducibility (less than +/- 3.6%), low blank values (60-80 cpm), speed (2 h per 30 assays), and capability to measure activity in small amounts of tissue (10-50 mg wet wt).     

Monovalent cation activation in Escherichia coli inosine 5'-monophosphate dehydrogenase

Inosine 5'-monophosphate dehydrogenase (IMPDH) catalyzes the oxidation of inosine 5'-monophosphate (IMP) to xanthosine 5'-monophosphate with the concomitant reduction of NAD to NADH. Escherichia coli IMPDH is activated by K(+), Rb(+), NH(+)(4), and Cs(+). K(+) activation is inhibited by Li(+), Na(+), Ca(2+), and Mg(2+). This inhibition is competitive versus K(+) at high K(+) concentrations, noncompetitive versus IMP, and competitive versus NAD. Thus monovalent cation activation is linked to the NAD site. K(+) increases the rate constant for the pre-steady-state burst of NADH production, possibly by increasing the affinity of NAD. Three mutant IMPDHs have been identified which increase the value of K(m) for K(+): Asp13Ala, Asp50Ala, and Glu469Ala. In contrast to wild type, both Asp13Ala and Glu469Ala are activated by all cations tested. Thus these mutations eliminate cation selectivity. Both Asp13 and Glu469 appear to interact with the K(+) binding site identified in Chinese hamster IMPDH. Like wild-type IMPDH, K(+) activation of Asp50Ala is inhibited by Li(+), Na(+), Ca(2+), and Mg(2+). However, this inhibition is noncompetitive with respect to K(+) and competitive with respect to both IMP and NAD. Asp50 interacts with residues that form a rigid wall in the IMP site; disruption of this wall would be expected to decrease IMP binding, and the defect could propagate to the proposed K(+) site. Alternatively, this mutation could uncover a second monovalent cation binding site.     

Biochemical analysis of the modular enzyme inosine 5'-monophosphate dehydrogenase.

Two prominent domains have been identified in the X-ray crystal structure of inosine-5'-monophosphate dehydrogenase (IMPDH), a core domain consisting of an alpha/beta barrel which contains the active site and an inserted subdomain whose structure is less well defined. The core domain encompassing amino acids 1-108 and 244-514 of wild-type human IMPDH (II) connected by the tetrapeptide linker Ile-Arg-Thr-Gly was expressed. The subdomain including amino acids 99-244 of human wild-type IMPDH (II) was expressed as a His-tagged fusion protein, where the His-tag was removable by enterokinase cleavage. These two proteins as well as wild-type human IMPDH (II), all proteins expressed in Escherichia coli, have been purified to apparent homogeneity. Both the wild-type and core domain proteins are tetrameric and have very similar enzymatic activities. In contrast, the subdomain migrates as a monomer or dimer on a gel filtration column and lacks enzymatic activity. Circular dichroism spectropolarimetry indicates that the core domain retains secondary structure very similar to full-length IMPDH, with 30% alpha-helix and 30% beta-sheet vs 33% alpha-helix and 29% beta-sheet for wild-type protein. Again, the subdomain protein is distinguished from both wild-type and core domain proteins by its content of secondary structure, with only 15% each of alpha-helix and beta-sheet. These studies demonstrate that the core domain of IMPDH expressed separately is both structurally intact and enzymatically active. The availability of the modules of IMPDH will aid in dissecting the architecture of this enzyme of the de novo purine nucleotide biosynthetic pathway, which is an important target for immunosuppressive and antiviral drugs.     

The CBS subdomain of inosine 5'-monophosphate dehydrogenase regulates purine nucleotide turnover

Inosine 5'-monophosphate dehydrogenase (IMPDH) catalyses the rate-limiting step in guanine nucleotide biosynthesis. IMPDH has an evolutionary conserved CBS subdomain of unknown function. The subdomain can be deleted without impairing the in vitro IMPDH catalytic activity and is the site for mutations associated with human retinitis pigmentosa. A guanine-prototrophic Escherichia coli strain, MP101, was constructed with the subdomain sequence deleted from the chromosomal gene for IMPDH. The ATP content was substantially elevated in MP101 whereas the GTP content was slighty reduced. The activities of IMPDH, adenylosuccinate synthetase and GMP reductase were two to threefold lower in MP101 crude extracts compared with the BW25113 wild-type strain. Guanine induced a threefold reduction in the MP101 ATP pool and a fourfold increase in the GTP pool within 10 min of addition to growing cells; this response does not result from the reduced IMPDH activity or starvation for guanylates. In vivo kinetic analysis using 14-C tracers and 33-P pulse-chasing revealed mutation-associated changes in purine nucleotide fluxes and turnover rates. We conclude that the CBS subdomain of IMPDH may coordinate the activities of the enzymes of purine nucleotide metabolism and is essential for maintaining the normal ATP and GTP pool sizes in E. coli .     

Species-specific inhibition of inosine 5'-monophosphate dehydrogenase by mycophenolic acid

IMPDH catalyzes the oxidation of IMP to XMP with the concomitant reduction of NAD(+) to NADH. This reaction is the rate-limiting step in de novo guanine nucleotide biosynthesis. Mycophenolic acid (MPA) is a potent inhibitor of mammalian IMPDHs but a poor inhibitor of microbial IMPDHs. MPA inhibits IMPDH by binding in the nicotinamide half of the dinucleotide site and trapping the covalent intermediate E-XMP. The MPA binding site of resistant IMPDH from the parasite Tritrichomonas foetuscontains two residues that differ from human IMPDH. Lys310 and Glu431 of T. foetus IMPDH are replaced by Arg and Gln, respectively, in the human type 2 enzyme. We characterized three mutants of T. foetusIMPDH: Lys310Arg, Glu431Gln, and Lys310Arg/Glu431Gln in order to determine if these substitutions account for the species selectivity of MPA. The mutation of Lys310Arg causes a 10-fold decrease in the K(i) for MPA inhibition and a 8-13-fold increase in the K(m) values for IMP and NAD(+). The mutation of Glu431Gln causes a 6-fold decrease in the K(i) for MPA. The double mutant displays a 20-fold increase in sensitivity to MPA. Pre-steady-state kinetics were performed to obtain rates of hydride transfer, NADH release, and hydrolysis of E-XMP for the mutant IMPDHs. The Lys310Arg mutation results in a 3-fold increase in the accumulation level of E-XMP, while the Glu431Gln mutation has only a minimal effect on the kinetic mechanism. These experiments show that 20 of the 450-fold difference in sensitivity between the T. foetus and human IMPDHs derive from the residues in the MPA binding site. Of this, 3-fold can be attributed to a change in kinetic mechanism. In addition, we measured MPA binding to enzyme adducts with 6-Cl-IMP and EICARMP. Neither of these adducts proved to be a good model for E-XMP.     

重组金黄色葡萄球菌次黄嘌呤单核苷酸脱氢酶与脂蛋白(a)的相互作用

次黄嘌呤单核苷酸脱氢酶(IMPDH)是金黄色葡萄球菌(S.aureus)表面的纤溶酶原(Plg)受体之一,它可以通过赖氨酸结合位点(LBS)与Plg相结合。脂蛋白(a)[Lp(a)]中的载脂蛋白(a)[Apo(a)]与Plg有很高的同源性,即两者的Kringle结构域都含有LBS,其中Apo(a)的KIV10含有强的LBS。因此本实验提出了Lp(a)应该能够与S.aureus表面的Plg受体相结合,进而可能竞争性抑制S.aureus与Plg结合的假说。本研究克隆了S.aureus的IMPDH基因,酶切后将其连接到表达载体pASK-IBA37中,并在大肠杆菌BL21中表达了该重组蛋白(rIMPDH)。通过酶联免疫吸附试验(ELISA)、亲和色谱层析及Western blot对rIMPDH与Lp(a)的相互作用机制进行了研究。结果表明rIMPDH可以通过LBS与Lp(a)和rKIV10发生特异性结合,而且一定浓度的赖氨酸类似物6-氨基己酸(EACA)可以抑制这种结合,然而本研究并未发现Lp(a)和rKIV10对rIMPDH与Plg的相互作用有明显的抑制。     

Targeted disruption of the inosine 5'-monophosphate dehydrogenase type I gene in mice

Inosine 5'-monophosphate dehydrogenase (IMPDH) is the critical, rate-limiting enzyme in the de novo biosynthesis pathway for guanine nucleotides. Two separate isoenzymes, designated IMPDH types I and II, contribute to IMPDH activity. An additional pathway salvages guanine through the activity of hypoxanthine-guanine phosphoribosyltransferase (HPRT) to supply the cell with guanine nucleotides. In order to better understand the relative contributions of IMPDH types I and II and HPRT to normal biological function, a mouse deficient in IMPDH type I was generated by standard gene-targeting techniques and bred to mice deficient in HPRT or heterozygous for IMPDH type II. T-cell activation in response to anti-CD3 plus anti-CD28 antibodies was significantly impaired in both single- and double-knockout mice, whereas a more general inhibition of proliferation in response to other T- and B-cell mitogens was observed only in mice deficient in both enzymes. In addition, IMPDH type I(-/-) HPRT(-/0) splenocytes showed reduced interleukin-4 production and impaired cytolytic activity after antibody activation, indicating an important role for guanine salvage in supplementing the de novo synthesis of guanine nucleotides. We conclude that both IMPDH and HPRT activities contribute to normal T-lymphocyte activation and function.     

A mutational analysis of the active site of human type II inosine 5'-monophosphate dehydrogenase

The oxidation of IMP to XMP is the rate-limiting step in the de novo synthesis of guanine ribonucleotides. This NAD-dependent reaction is catalyzed by the enzyme inosine monophosphate dehydrogenase (IMPDH). Based upon the recent structural determination of IMPDH complexed to oxidized IMP (XMP*) and the potent uncompetitive inhibitor mycophenolic acid (MPA), we have selected active site residues and prepared mutants of human type II IMPDH. The catalytic parameters of these mutants were determined. Mutations G326A, D364A, and the active site nucleophile C331A all abolish enzyme activity to less than 0.1% of wild type. These residues line the IMP binding pocket and are necessary for correct positioning of the substrate, Asp364 serving to anchor the ribose ring of the nucleotide. In the MPA/NAD binding site, significant loss of activity was seen by mutation of any residue of the triad Arg322, Asn303, Asp274 which form a hydrogen bonding network lining one side of this pocket. From a model of NAD bound to the active site consistent with the mutational data, we propose that these resides are important in binding the ribose ring of the nicotinamide substrate. Additionally, mutations in the pair Thr333, Gln441, which lies close to the xanthine ring, cause a significant drop in the catalytic activity of IMPDH. It is proposed that these residues serve to deliver the catalytic water molecule required for hydrolysis of the cysteine-bound XMP* intermediate formed after oxidation by NAD.     

Crystal structure of a ternary complex of Tritrichomonas foetus inosine 5'-monophosphate dehydrogenase: NAD+ orients the active site loop for catalysis

Inosine 5'-monophosphate dehydrogenase (IMPDH) catalyzes the conversion of IMP to XMP with the reduction of NAD(+), which is the rate-limiting step in the biosynthesis of guanine nucleotides. IMPDH is a promising target for chemotherapy. Microbial IMPDHs differ from mammalian enzymes in their lower affinity for inhibitors such as mycophenolic acid (MPA) and thiazole-4-carboxamide adenine dinucleotide (TAD). Part of this resistance is determined by the coupling between nicotinamide and adenosine subsites in the NAD(+) binding site that is postulated to involve an active site flap. To understand the structural basis of the drug selectivity, we solved the X-ray crystal structure of the catalytic core domain of Tritrichomonas foetus IMPDH in complex with IMP and beta-methylene-TAD at 2.2 A resolution. Unlike previous structures of this enzyme, the active site loop is ordered in this complex, and the catalytic Cys319 is 3.6 A from IMP, in the same plane as the hypoxanthine ring. The active site loop forms hydrogen bonds to the carboxamide of beta-Me-TAD which suggests that NAD(+) promotes the nucleophillic attack of Cys319 on IMP. The interactions of the adenosine end of TAD are very different from those in the human enzyme, suggesting the NAD(+) site may be an exploitable target for the design of antimicrobial drugs. In addition, a new K(+) site is observed at the subunit interface. This site is adjacent to beta-Me-TAD, consistent with the link between the K(+) activation and NAD(+). However, contrary to the coupling model, the flap does not cover the adenosine subsite and remains largely disordered.     

Retinal isoforms of inosine 5'-monophosphate dehydrogenase type 1 are poor nucleic acid binding proteins

The RP 10 form of autosomal dominant retinitis pigmentosa (adRP) is caused by mutations in the widely expressed protein inosine 5′-monophosphate dehydrogenase type 1 (IMPDH1). These mutations have no effect on the enzymatic activity of IMPDH1, but do perturb the association of IMPDH1 with nucleic acids. Two newly discovered retinal-specific isoforms, IMPDH1(546) and IMPDH1(595), may provide the key to the photoreceptor specificity of disease [S.J. Bowne, Q. Liu, L.S. Sullivan, J. Zhu, C.J. Spellicy, C.B. Rickman, E.A. Pierce, S.P. Daiger, Invest. Ophthalmol. Vis. Sci. 47 (2006) 3754-3765]. Here we express and characterize the normal IMPDH1(546) and IMPDH1(595), together with their adRP-linked variants, D226N. The enzymatic activity of the purified IMPDH1(546), IMPDH1(595) and the D226N variants is indistinguishable from the canonical form. The intracellular distribution of IMPDH1(546) and IMPDH1(595) is also similar to the canonical IMPDH1 and unaffected by the D226N mutation. However, unlike the canonical IMPDH1, the retinal specific isoforms do not bind significant fractions of a random pool of oligonucleotides. This observation indicates that the C-terminal extension unique to the retinal isoforms blocks the nucleic acid binding site of IMPDH1, and thus uniquely regulates protein function within photoreceptors.     

Differential splicing of Pneumocystis carinii f. sp. carinii inosine 5'-monophosphate dehydrogenase pre-mRNA

Inosine 5'-monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme in guanine nucleotide metabolism that has drawn attention as a drug target in several organisms. Pneumocystis carinii f. sp. carinii IMPDH mRNA (GeneBank Accession No: U42442) previously identified from cultured organisms yielded a predicted amino acid sequence about 70 amino acids shorter at the amino terminus than IMPDH from other species. Recent research has shown that the amino terminal region is important for enzyme activity, suggesting that the previous putative P. carinii IMPDH might not represent full length, functional enzyme. To test this hypothesis, RT-PCR was performed with total RNA isolated from P. carinii f. sp. carinii. Three IMPDH splicing variants were found and splicing preference was observed: P. carinii isolated from infected rat lung contained primarily splicing variant one (introns two and four deleted), but organisms from spinner flask culture contained primarily splicing variant three (all four introns deleted). Importantly, splicing variant one (GeneBank Accession No: AF196975) contained an open reading frame for 529 amino acids, a size comparable to that of other eukaryotic IMPDH forms. The other variants contained the same open reading frame (454 amino acids) previously reported. Sequence analysis and complementation studies suggest variant one represents the full length, catalytically active form of P. carinii IMPDH. The differential splicing of the enzyme may reflect a mechanism by which the organism regulates the expression of IMPDH in response to environmental stresses.     

Crystal structure at 2.4 A resolution of Borrelia burgdorferi inosine 5'-monophosphate dehydrogenase: evidence of a substrate-induced hinged-lid motion by loop 6

The conversion of inosine 5'-monophosphate (IMP) to xanthosine 5'-monophosphate (XMP) is the committed and rate-limiting reaction in de novo guanine nucleotide biosynthesis. Inosine 5'- monophosphate dehydrogenase (IMPDH) is the enzyme that catalyzes the oxidation of IMP to XMP with the concomitant reduction of nicotinamide adenine dinucleotide (from NAD(+) to NADH). Because of its critical role in purine biosynthesis, IMPDH is a drug design target for anticancer, antiinfective, and immunosuppressive chemotherapy. We have determined the crystal structure of IMPDH from Borrelia burgdorferi, the bacterial spirochete that causes Lyme disease, with a sulfate ion bound in the IMP phosphate binding site. This is the first structure of IMPDH in the absence of substrate or cofactor where the active-site loop (loop 6), which contains the essential catalytic residue Cys 229, is clearly defined in the electron density. We report that a seven residue region of loop 6, including Cys229, is tilted more than 6 A away from its position in substrate- or substrate analogue-bound structures of IMPDH, suggestive of a conformational change. The location of this loop between beta6 and alpha6 links IMPDH to a family of beta/alpha barrel enzymes known to utilize this loop as a functional lid during catalysis. Least-squares minimization, root-mean-square deviation analysis, and inspection of the molecular surface of the loop 6 region in the substrate-free B. burgdorferi IMPDH and XMP-bound Chinese hamster IMPDH show that loop 6 follows a similar pattern of hinged rigid-body motion and indicates that IMPDH may be using loop 6 to bind and sequester substrate and to recruit an essential catalytic residue.     

Purification and specificity of antibodies to inosine 5'-monophosphate

Antibodies to inosine 5'-monophosphate elicited in rabbits by immunization with a conjugate of IMP (oxidized with periodate) and bovine serum albumin have been purified by affinity chromatography. By the use of two affinity columns, Sepharose-IMP and Sepharose-oligo(I), the antibodies have been fractionated into three fractions. By gel diffusion, the three fractions were found to react with the conjugates of bovine serum albumin and IMP, GMP and AMP respectively. The association constants for the binding of the Fab fragments purified on the Sepharose-oligo(I) column and several haptens have been deduced from fluorescence experiments. It is shown that the base and the phosphate group play an important part in the binding of IMP to Fab fragments. No reaction has been found between the antibodies and poly(I).poly(C) by gel diffusion. However, the antibodies interact with poly(I).poly(C) since they decrease the thermal stability of poly(I).poly(C).     

3-Hydrogenkwadaphnin targets inosine 5'-monophosphate dehydrogenase and triggers post-G1 arrest apoptosis in human leukemia cell lines

3-Hydrogenkwadaphnin (3-HK) is a recently characterized daphnane-type compound isolated from Dendrostellera lessertii with high anti-tumor activity in animal models. Herein, we report on time- and dose-dependent effects of this compound on growth, differentiation, IMPDH inhibition, cell cycle and apoptosis of a panel of human leukemia cell lines (HL-60, K562 and Molt4). The drug decreased the growth of leukemia cells in less than 24 h of treatment. However, longer exposure times and/or higher concentrations were required to promote cell apoptosis. Cell cycle analysis revealed the accumulation of cells in their G1 phase as early as 12 h after drug exposure but sub-G1 population was recorded after 24 h. Occurrence of apoptosis was constantly accompanied by morphological (staining with DNA-binding dyes) and biochemical (DNA fragments) variations among drug-treated cells. Despite these observations, non-activated normal human PBL were insensitive to the drug action. In addition, treatment of PHA-activated PBL, K562, Molt4 and HL-60 cells with a single dose of the drug for 24 h led to the inhibition of IMPDH activity by almost 37, 38, 44 and 50%, respectively. In contrast, no difference in IMPDH activities were seen between normal PBL and the drug treated PBL cells. Restoration of the depleted GTP concentration by exogenous addition of guanosine (25-50 microM) reversed the drug effects on cell growth, DNA fragmentation and apoptosis. Furthermore, the drug effects were potentiated by exogenous addition of hypoxanthine to the drug-treated cells. Reduction of the drug potency on the non-proliferative (retinoic acid treated) HL-60 cells by almost 40%, compared to the proliferative cells, clearly shows type II IMPDH as one of the main targets of the drug. These results suggest that 3-HK may be a powerful candidate for treatment of leukemia.     

An enzymatic method for inosine 5'-monophosphate in the femtomole range

A method for measuring inosine 5'-monophosphate (IMP) by enzymatic generation of NADPH is described. Procedures are given for direct fluorometric assay in the nanomole range and indirect measurement with amplification by enzymatic cycling in the pico- and femtomole ranges. The most sensitive procedure represents a nearly 50,000-fold increase in sensitivity over enzymatic methods now available. Specificity of the assay was greatly enhanced by the use of the antibiotic coformycin, a potent inhibitor of adenosine deaminase (EC 3.5.4.4). This enzyme was found to be a major contaminant of one of the necessary enzymes, phosphoglucomutase (EC 2.7.5.1). The use of the method is illustrated by measurements of IMP in single stimulated and control rat muscle fibers.     

Validation and application of a high-performance liquid chromatographic-based assay for determination of the inosine 5'-monophosphate dehydrogenase activity in erythrocytes

Thiopurine drug monitoring has become an important issue in treating children with acute lymphoblastic leukaemia (ALL). In this population, a genetic polymorphism causes wide differences in the activity of thiopurine S-methyletransferase (TPMT)--the rate-limiting enzyme of the thiopurine degradation metabolism--leading to the necessity of drug dose adjustments. It is not yet known if similar differences exist in the inosine 5'-monophosphate dehydrogenase (IMPDH; EC 1.1.1.205), the rate-limiting enzyme of the thiopurine synthesis. To test this, we established and validated a high-performance liquid chromatographic (HPLC)-based assay to determine the IMPDH enzyme activity in erythrocytes. The remarkable features of this assay are its simple erythrocyte separation/haemolysis and assay conditions and a distinct segregation of xanthosine 5'-monophosphate (XMP) from the clear supernatant after precipitation. The probes were processed without a time-consuming extraction and heating procedure and the assay demonstrated a good intra- and interday stability as well as a recovery rate of approximately 100%. The IMPDH enzyme activity was measured in erythrocytes of 75 children with diagnosis of ALL before starting antileukaemic therapy and their activity compared to those of 35 healthy adult controls. The measured enzyme activity was wide ranging in both groups. The individual enzyme activity differences observed in children with ALL might led to differences in the thionucleotide levels in those undergoing the standard thiopurine dose regimen.     

Inhibition of inosine monophosphate dehydrogenase (IMPDH) by 2-[2-(Z)-fluorovinyl]inosine 5'-monophosphate

Inosine 5'-monophosphate dehydrogenase (IMPDH; EC 1.1.1.205) isolated from Escherichia coli B3 cells was strongly inhibited by 2-[2-(Z)-fluorovinyl]inosine 5'-monophosphate (2-FVIMP). Inhibition of IMPDH appears to be irreversible with k(inact) and K(i) values of 0.0269 s(-1) and 1.11 microM, respectively.     

Participation of yeast inosine 5'-monophosphate dehydrogenase in an in vitro complex with a fragment of the C-rich telomeric strand

As part of our investigation of the i-motif, an intercalated structure formed by C-rich nucleic acid sequences, we searched for proteins of Saccharomyces cerevisiae which could associate with a sequence of the C-rich telomeric strand, d((CCCACA)(3)CCC). A gel retardation assay of yeast protein extract, in conditions where the DNA fragment folds into an intramolecular i-motif, shows formation of one major retarded band. The retarding factor was further characterized by a differential affinity procedure using streptavidin beads coated (or not coated) with the biotin-labeled DNA fragment. Differentially bound proteins were isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and identified by mass spectroscopy and Edman degradation as Imd2p, Imd3p and Imd4p. These highly similar (>95%) proteins are analogs of the two human NAD-dependent inosine 5'-monophosphate dehydrogenases (IMPDH) which occur as tetramers. The mass of the protein, as determined by gel exclusion chromatography, is about 250 kDa and is compatible with an IMPDH tetramer, but other compositions, involving non-IMPDH components, are not excluded. We note that the genes coding for Imd2p and Imd3p are located close to the telomere, and could therefore be subject to silencing by the telomere position effect.