Triazole-linked inhibitors of inosine monophosphate dehydrogenase from human and Mycobacterium tuberculosis.

The modular nature of nicotinamide adenine dinucleotide (NAD)-mimicking inosine monophsophate dehydrogenase (IMPDH) inhibitors has prompted us to investigate novel mycophenolic adenine dinucleotides (MAD) in which 1,2,3-triazole linkers were incorporated as isosteric replacements of the pyrophosphate linker. Synthesis and evaluation of these inhibitors led to identification of low nanomolar inhibitors of human IMPDH and more importantly the first potent inhibitor of IMPDH from Mycobacterium tuberculosis (mtIMPDH). Computational studies of these IMPDH enzymes helped rationalize the observed structure-activity relationships. Additionally, the first cloning, expression, purification and characterization of mtIMPDH is reported.

Identification and characterization of inosine 5-monophosphate dehydrogenase in Streptococcus suis type 2.

Streptococcus suis type 2 is a swine pathogen responsible for diverse diseases. Although many virulent factors have been identified and studied, relatively little is known about the pathogenic mechanisms of type 2. The aim of the study was to identify and understand the characterization of Inosine 5-monophosphate dehydrogenase (IMPDH). A 957-bp gene, impdh, was identified in the virulent S. suis serotype 2 (SS2), and analysis of the predicted IMPDH sequence revealed IMP dehydrogenase/GMP reductase domain. The gene encoding for the IMPDH of S. suis was cloned and sequenced. The DNA sequence contained an open reading frame encoding for a 318 amino acid polypeptide exhibiting 23% sequence identity with the IMPDH from Streptococcus pyogenes (YP281355) and Streptococcus pneumoniae (ZP00404150). Using the pET(32) expression plasmid, the impdh gene was inducibly overexpressed in Escherichia coli to produce IMPDH with a hexahistidyl N-terminus to permit its purification. The (His)6 IMPDH protein was found to possess functional IMPDH enzymatic activity after the purification. The impdh-knockout SS2 mutant ( Delta IMPDH) constructed in this study was slower in growth and one pH unit higher than SS2-H after 6 h of culturing, and found to be attenuated in mouse models of infection for 2.5 times and not be capable of causing death in porcine models of infection in contrast with the parent SS2-H.

Specific DNA binding of a potential transcriptional regulator, inosine 5'-monophosphate dehydrogenase-related protein VII, to the promoter region of a methyl coenzyme m reductase I-encoding operon retrieved from Methanothermobacter thermautotrophicus strain DeltaH.

Two methyl coenzyme M reductases (MCRs) encoded by the mcr and mrt operons of the hydrogenotrophic methanogen Methanothermobacter thermautotrophicus DeltaH are expressed in response to H(2) availability. In the present study, cis elements and trans-acting factors responsible for the gene expression of MCRs were investigated by using electrophoretic mobility shift assay (EMSA) and affinity particle purification. A survey of their operator regions by EMSA with protein extracts from mrt-expressing cultures restricted them to 46- and 41-bp-long mcr and mrt upstream regions, respectively. Affinity particle purification of DNA-binding proteins conjugated with putative operator regions resulted in the retrieval of a protein attributed to IMP dehydrogenase-related protein VII (IMPDH VII). IMPDH VII is predicted to have a winged helix-turn-helix DNA-binding motif and two cystathionine beta-synthase domains, and it has been suspected to be an energy-sensing module. EMSA with oligonucleotide probes with unusual sequences showed that the binding site of IMPDH VII mostly overlaps the factor B-responsible element-TATA box of the mcr operon. The results presented here suggest that IMPDH VII encoded by MTH126 is a plausible candidate for the transcriptional regulator of the mcr operon in this methanogen.

Selective inhibition of human Molt-4 leukemia type II inosine 5'-monophosphate dehydrogenase by the 1,5-diazabicyclo[3.1.0]hexane-2,4-diones.

Inosine 5'-monophosphate dehydrogenase (IMPDH) is the rate-limiting enzyme in de novo purine biosynthesis. IMPDH activity results from expression of two isoforms. Type I is constitutively expressed and predominates in normal resting cells, while Type II is selectively up-regulated in neoplastic and replicating cells. Inhibitors of IMPDH activity selectively targeting the Type II isoform have great potential as cancer chemotherapeutic agents. For this study, an expression system was developed which yields 35-50 mg of soluble, purified recombinant Type I and II protein from 1 L of bacteria. In addition, three 1,5-diazabicyclo[3.1.0]hexane-2,4-diones were synthesized and shown to act as specific inhibitors of human recombinant Type II IMPDH. The agents are competitive inhibitors with respect to the endogenous substrate IMP and K(i) values range from 5 to 44 microM but were inactive as inhibitors of the Type I isoform at concentrations ranging from 0.5 to 500 microM. IC(50) values for recombinant Type II inhibition were determined and compared to IC(50) values obtained from Molt-4 cell extracts of IMPDH. Cytotoxicity assays revealed that the compounds inhibited Molt-4 leukemia growth with ED(50) values of 3.2-7.6 microM. Computational docking studies predict that the compounds bind to IMPDH in the IMP-binding site, although interactions with residues differ from those previously determined to interact with bound IMP. While all residues predicted to interact directly with the bound compounds are conserved in the Type I and Type II isoforms, sequence divergence within a helix adjacent to the active site may contribute to the observed selectivity for the human Type II isoform. These compounds represent the first class of selective IMPDH Type II inhibitors which may serve as lead compounds for the development of isoform-selective cancer chemotherapy.

Identification of the IMP binding site in the IMP dehydrogenase from Tritrichomonas foetus.

The IMP dehydrogenase from Tritrichomonas foetus has been identified as a potential target for antitritrichomonial chemotherapy. The gene encoding this enzyme was expressed in transformed Escherichia coli, and the recombinant protein was purified to homogeneity with an average yield of 3 mg of protein per liter of bacterial culture. Kinetic characterizations verified that the recombinant enzyme is in the authentic native state. 6-Cl-IMP, an irreversible inhibitor of the enzyme, was found to protect cysteine residue 319 of the enzyme against carboxymethylation by iodoacetamide. Radiolabeled IMP was covalently bound to the enzyme during the enzyme-catalyzed reaction via the formation of a specific adduct with cysteine residue 319. It is thus postulated that the conversion of IMP to XMP catalyzed by the IMP dehydrogenase from T. foetus is mediated by a nucleophilic attack of cysteine-319 in the enzyme protein to IMP at, most likely, its 2-position to facilitate a hydride transfer to NAD, resulting in the formation of a covalent intermediate between substrate and enzyme.

Recombinant human inosine monophosphate dehydrogenase type I and type II proteins. Purification and characterization of inhibitor binding.

Inosine monophosphate dehydrogenase (IMPDH) activity results from the expression of two separate genes, and the resulting proteins (type I and type II) are 84% identical at the amino acid level. Although the type II mRNA is expressed at higher levels in proliferating cells, both mRNAs, and by extrapolation both proteins, are present in normal and malignant cells. Since IMPDH is an important target for the development of drugs with both chemotherapeutic and immunosuppressive activity, we have compared the kinetic and physical properties of the two human enzymes expressed in and purified from Escherichia coli. Type I and II IMPDH had kcat values of 1.8 and 1.4 sec-1, respectively, with Km values for IMP of 14 and 9 microM and Km values for NAD of 42 and 32 microM. The two enzymes were inhibited competitively by the immunosuppressive agent mizoribine 5'-monophosphate (MMP) with Ki values of 8 and 4 nM and inhibited uncompetitively by mycophenolic acid with Ki values of 11 and 6 nM. The association of MMP to either isozyme, as monitored by fluorescence quenching, was relatively slow with kon values of 3-8 x 10(4) M-1 sec-1 and koff values of 3 x 10(-4) sec-1 (half-lives of 36-43 min). Thus, MMP is a potent, tight-binding competitive inhibitor that does not discriminate between the two IMPDH isozymes.

Cloning, sequencing, and structural analysis of the DNA encoding inosine monophosphate dehydrogenase (EC 1.1.1.205) from Tritrichomonas foetus.

The inosine monophosphate dehydrogenase (IMPDH) of the parasitic protozoan Tritrichomonas foetus is a purine salvage enzyme with a subunit molecular weight of 58,000. The enzyme has been purified to homogeneity by Verham et al. (Molecular and Biochemical Parasitology 24, 1-12, 1987) and characterized in more detail by Hedstrom and Wang (Biochemistry 29, 849-854, 1990). We used a polyclonal antibody directed against the purified enzyme to identify three cDNA clones from T. foetus. These clones were sequenced and found to contain an open reading frame encoding 497 amino acids. By complementation studies on an Escherichia coli mutant with its IMPDH gene deleted, the cDNA clones were able to transform the bacterial cells to grow on minimal medium without guanine. One of the cDNA clones, 2aa1, was used to identify two genomic clones, 2d1c and 3m4b, both containing a 4.1-kb HindIII fragment. The fragment was subcloned into the Bluescript KS+ plasmid, sequenced, and found to contain the same open reading frame as the cDNA clone except that it encodes six additional amino acid residues at the N-terminus. Its sequence has a 34% identity with that of the human IMPDH, 32% with that of E. coli IMPDH, and 31% with that of Leishmania donovani IMPDH. The molecular weight of the deduced protein is 55,534. Two segments of polypeptide that are conserved in all other IMPDHs, containing the putative NAD+ and IMP binding sites, are also relatively conserved in T. foetus. Since the parasite enzyme differs from the bacterial and mammalian IMPDHs by a very high Km value for NAD+ and an even higher KI value for mycophenolic acid (MPA) (Verham et al. 1987; Hedstrom and Wang 1990), the sequence of the parasite enzyme may provide information on the mechanism of MPA binding and the chance for other specific inhibitor design.

Characterization of human type I and type II IMP dehydrogenases.

Human IMP dehydrogenase, a target for anticancer and immunosuppressive chemotherapy, exists as two isoforms, types I and II. Nonfusion sequences of each isoform were overexpressed in an IMP dehydrogenase-deficient strain of Escherichia coli and purified to homogeneity. Both recombinant isoforms were tetramers, which was in agreement with the subunit structure of the native mammalian enzyme. The results of initial velocity and product inhibition studies were consistent with an Ordered Bi Bi kinetic mechanism for both isoforms. Substrate affinities were similar for types I and II with Km values of 18 and 9.3 microM, respectively, for IMP, and 46 and 32 microM, respectively, for NAD.kcat values were 1.5 and 1.3 s-1 at 37 degrees C for types I and II, respectively. Xanthosine 5'-monophosphate and NADH inhibited the two isoforms with identical inhibition patterns and inhibition constants. Mycophenolic acid, however, inhibited the type II enzyme with a 4.8-fold lower K than the type I. Selective inhibitors of the inducible type II isoform may mitigate toxicity caused by inhibition of the constitutively expressed type I isoform.

Inosine monophosphate dehydrogenase from porcine (Sus scrofa domestica) thymus: purification and properties.

1. IMP dehydrogenase (EC 1.1.1.205) from porcine thymus glands has been purified to homogeneity. 2. The enzyme has a subunit MW of 57 kDa and an amino acid composition similar to those obtained from other normal and cancerous mammalian cells. 3. The apparent Km values at pH 8.0 for IMP and NAD+ are 7 and 16 microM, respectively. 4. GMP, XMP and AMP are competitive inhibitors towards IMP and Ki values of 50, 85 and 282 microM, respectively. 5. The effectiveness of nucleotides to protect inactivation by CI-IMP is IMP > GMP > XMP > AMP.

Expression of human IMP dehydrogenase types I and II in Escherichia coli and distribution in human normal lymphocytes and leukemic cell lines.

Two distinct cDNAs encoding proteins with 84% sequence identity have been isolated for human IMP dehydrogenase (EC 1.1.1.205) (Natsumeda, Y., Ohno, S., Kawasaki, H., Konno, Y., Weber, G., and Suzuki, K. (1990) J. Biol. Chem. 265, 5292-5295), an important target in antileukemic chemotherapy. We constructed expression plasmids containing these cDNAs in full length with pUC plasmids and produced lacZ'-IMP dehydrogenase fusion proteins in Escherichia coli. Both synthesized proteins exhibited IMP dehydrogenase activity and were partially separated from endogenous E. coli IMP dehydrogenase. By injecting the fusion proteins into mice we generated a polyclonal antibody specific to type I IMP dehydrogenase and an antibody which reacted with both types. Immunoblot analysis revealed that the total amounts of types I and II enzymes increased in human leukemic cell lines K562 and HL-60 in agreement with the increase in IMP dehydrogenase activity to 7.8- and 9.4-fold, respectively, above that of normal lymphocytes. The extent of expression of type I IMP dehydrogenase was similar in these cells, however, indicating that the increase in IMP dehydrogenase amount in leukemic cells was due to specific up-regulation of type II enzyme. Northern blot analysis also showed specific and predominant expression of type II in the leukemic cells. Thus, de novo GTP biosynthesis may be controlled differently in normal and neoplastic cells by different IMP dehydrogenase molecular species.

IMP dehydrogenase and action of antimetabolites in human cultured blast cells.

Tiazofurin was demonstrated to be an effective inhibitor of the growth of human cultured blast cells, and the high specific activities of IMP dehydrogenase (EC 1.1.1.205) were observed in all the cell extracts tested. IMP dehydrogenase has been purified to homogeneity from MOLT 4F human T-lymphoblast, and the Km values for IMP and NAD were 29 and 54 microM, respectively. The inhibitory mechanisms of thiazole-4-carboxamide adenine dinucleotide (TAD) and ribavirin 5'-monophosphate (RMP), the active forms of the antimetabolites tiazofurin and ribavirin, were investigated on the purified enzyme. RMP inhibits competitively with respect to IMP as well as XMP, and the inhibition by TAD was similar to that by NADH, which was uncompetitive with NAD. However, the Ki values of RMP (0.58 microM) and TAD (0.075 microM) were several orders of magnitude lower than those of XMP (85 microM) and NADH (94 microM). Thus, the drugs interact with the two distinct sites of IMP dehydrogenase with much higher affinities than the natural substrates and products. Preincubation of the purified enzyme with RMP enhanced its inhibitory effect in a time-dependent manner, and the enhancement was further increased by the addition of TAD. The combination of tiazofurin and ribavirin exerted a synergistic effect on the growth inhibition in MOLT 4F cells.

Increased activity, amount, and altered kinetic properties of IMP dehydrogenase from mycophenolic acid-resistant neuroblastoma cells.

Mouse wild-type neuroblastoma cells (NB cells) were stepwise selected for 10,000-fold increased resistance to mycophenolic acid (NB-Myco cells), an inhibitor of IMP dehydrogenase (IMP:NAD+ oxidoreductase, EC 1.1.1.205). IMP dehydrogenase activity was increased 25-fold, from 3.1 to 75 nmol/min.mg of protein; and a 56.7-kDa peptide was increased in abundance 200-500-fold in NB-Myco as compared to NB cells. Purification and sequence analysis confirmed that the abundant protein was IMP dehydrogenase. The stepwise selection, increased activity and protein abundance, and unstable phenotype are indirect evidence for a process of gene amplification. Kinetic findings consistent with an Ordered Bi Bi mechanism were indicative of IMP dehydrogenase having undergone mutation. The Michaelis constants were unchanged for IMP (14 and 13 microM) and increased 4-fold for NAD from 25 to 94 microM for NB and NB-Myco cells, respectively. The Ki for mycophenolic acid was increased 2400-fold from 1.4 nM to 3.4 microM for the enzyme from NB versus NB-Myco cells, and the Ki for XMP was increased 4-fold from 78 to 336 microM. Mycophenolic acid exhibited uncompetitive inhibition with IMP, consistent with the formation of a dead end E-XMP-inhibitor complex. The cellular GTP concentration was increased 2-fold in resistant cells and, upon removal of mycophenolic acid, further increased to 4.5-fold that of NB cells.

Action of the active metabolites of tiazofurin and ribavirin on purified IMP dehydrogenase.

The inhibitory mechanisms of ribavirin 5'-monophosphate (RMP) and thiazole-4-carboxamide adenine dinucleotide (TAD), the active forms of the antimetabolites ribavirin and tiazofurin, were investigated in IMP dehydrogenase purified to homogeneity from rat hepatoma 3924A. The hepatoma IMP dehydrogenase has a tetrameric structure with a subunit molecular weight of 60,000. For the substrates IMP and NAD+, Km's were 23 and 65 microM, respectively. Product-inhibition patterns showed an ordered Bi-Bi mechanism for the enzyme reaction where IMP binds to the enzyme first, followed by NAD+; NADH dissociates from the ternary complex first and then XMP is released. XMP interacts with the free enzyme and competes for the ligand site with IMP, while NADH binds to the enzyme-XMP complex. RMP exerted the same inhibitory mechanisms as XMP, and the inhibition by TAD was similar to that by NADH. However, the Ki values for RMP (0.8 microM) and TAD (0.13 microM) were orders of magnitude lower than those of XMP (136 microM) and NADH (210 microM). Thus, the drugs interact with IMP dehydrogenase with higher affinities than the natural substrates and products, RMP with the IMP-XMP site and TAD with the NADH site. Preincubation of the purified enzyme with RMP enhanced its inhibitory effect in a time-dependent manner. The enzyme was protected from this inactivation by IMP or XMP. These results provide a biochemical basis for combination chemotherapy with tiazofurin and ribavirin targeted against the two different ligand sites of IMP dehydrogenase.

Inhibition of inosine monophosphate dehydrogenase by sesquiterpene lactones.

Inosine monophosphate (IMP) dehydrogenase had previously been determined to be a likely target enzyme for the sesquiterpene lactones, a class of potential anti-neoplastic drugs. IMP dehydrogenase was purified approx. 770-fold from the P-388 lymphocytic leukemia tumor cell line. The Km values for the substrates, IMP and NAD, were determined to be 12 microM and 25 microM, respectively. Xanthine monophosphate (XMP) was shown to be a competitive inhibitor with a Ki of 67 microM. Mycophenolic acid gave mixed-type inhibition with a Ki of 8 nM for the noncompetitive component and a Ki of 2 nM for the competitive component. Dissociation constants (Kd) and rate constants for inhibition of IMP dehydrogenase by nine different sesquiterpene lactones were determined. The highest Kd was seen with 2,3-dihydrohelenalin while the lowest Kd was observed with bis-helenalinyl malonate. Binding of the drugs by IMP dehydrogenase increased as the size of the drug increased. Also, changes in structure at position 6 had a relatively large effect on the Kd. There was no correlation with hydrophobicity, as determined by octanol/water partition. The first-order rate constants for the reaction of the sesquiterpene lactones with IMP dehydrogenase (k1) and the second-order rate constants for the reaction of the sesquiterpene lactones with glutathione (k2) were also determined. The rate constants for most of the sesquiterpene lactones with the alpha-methylene-gamma-lactone moiety were similar and were approximately twice as great as the rate constants for those sesquiterpene lactones with only the alpha, beta-unsaturated cyclopentenone ring. Microlenin had approximately 5-times the reactivity of the other sesquiterpene lactones towards IMP dehydrogenase, but had approximately the same reactivity towards glutathione, suggesting that it was bound to the enzyme in a way which facilitated its reaction with one or more essential sulfhydryls. The same procedure was used for a series of N-substituted maleimide compounds with the N-substituent ranging in size from a methyl group to a benzyl group. The binding of the maleimide compounds was generally tighter than for the sesquiterpene lactones and there was an increase in binding with size.

Purification of IMP dehydrogenase from rat hepatoma 3924A.

IMP dehydrogenase (EC 1.1.1.205), the rate-limiting enzyme of de novo GTP biosynthesis and a promising target for cancer chemotherapy, was purified 4860-fold to homogeneity from rat hepatoma 3924A by a method including affinity chromatography in which IMP is bound to epoxy-activated Sepharose 6B. This affinity gel provided a specific elution of the enzyme with 0.5 mM IMP. The final enzyme preparation gave a single band with a molecular weight of 60,000 +/- 1000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis.

Purification, characterization, and kinetic analysis of inosine 5'-monophosphate dehydrogenase of Tritrichomonas foetus.

The IMP dehydrogenase of Tritrichomonas foetus, a parasitic protozoan incapable of de novo biosynthesis of purine nucleotides, has been purified about 1000-fold to apparent homogeneity. The purified enzyme demonstrated a 20-fold higher substrate turnover rate than the pure IMP dehydrogenase from sarcoma ascites tumor cells. It has a subunit molecular weight of 58,000, aggregates to a size of 380,000 at low ionic strength, and partly dissociates to a molecular weight of 270,000 in high salt concentrations. Unlike the IMP dehydrogenase of bacteria and mammals, the T. foetus enzyme does not require K+ for activity. The analysis of initial velocity and product inhibition data is consistent with a sequential, ordered bi bi kinetic mechanism for the parasite enzyme-catalyzed reaction, in which IMP binds before NAD+ and NADH is released before XMP. This is in contrast to the partially random mechanism of the bacterial enzyme which involves the formation of an enzyme-K+-(IMP) complex. Mycophenolic acid inhibits T. foetus IMP dehydrogenase uncompetitively versus both IMP and NAD+ with an apparent Ki of 9 microM. This value, which is several hundred-fold higher than that for mammalian IMP dehydrogenase, suggests significantly different binding properties of the mycophenolic acid site in T. foetus IMP dehydrogenase, which might be amenable to specific inhibitor design.

Conversion of 6-mercaptopurine to 6-thioguanylic acid in L-1210 cells and human leukemia cells.

The metabolism of 6-mercaptopurine (6-MP) in L-1210 mouse leukemia cells and human chronic myelocytic leukemia cells (CML cells) was examined. The acid-soluble fractions obtained from cells incubated with [8-14C]6-MP were chromatographed on a Dowex-1 formate resin column using a formic acid linear gradient elution system. Chromatography of the extract of L-1210 cells revealed four principal radioactive peaks. The fraction containing the third peak was hydrolyzed by snake venom 5'-nucleotidase (Crotalus adamanteus). Cellulose thin layer chromatography revealed that the radioactive peak of the hydrolysate corresponded to 6-thioguanosine. The results showed that 6-MP was converted to 6-thioinosinic acid (6-TIMP) and 6-thioguanylic acid (6-TGMP) in L-1210 cells. In order to elucidate the pathway of 6-MP conversion to 6-TGMP, we examined the interaction of [8-14C]6-TIMP and purified IMP dehydrogenase. It was found by DEAE-cellulose thin layer chromatography that the IMP dehydrogenase converted 6-TIMP to 6-thioxanthylic acid (6-TXMP). Dowex-1 chromatography of the acid-soluble extract of human CML cells incubated with [8-14C]-6-MP also revealed a radioactive peak corresponding to 6-TGMP. These results suggest that 6-MP is metabolized to 6-TGMP by serial conversion to 6-TIMP and 6-TXMP through the de novo GMP synthetic pathway in L-1210 cells and human CML cells.

IMP dehydrogenase. II. Purification and properties of the enzyme from Yoshida sarcoma ascites tumor cells.

The preceding paper showed that IMP dehydrogenase [IMP:NAD+ oxidoreductase, EC 1.2.1.14] tended to form a precipitable complex(es) through ionic and hydrophobic interactions. On the basis of these observations, a method was developed for purification of IMP dehydrogenase from Yoshida sarcoma ascites cells. On SDS-polyacrylamide gel electrophoresis, the purified preparation (1.19 U/mg protein) appeared homogeneous and its minimum molecular weight was estimated to be 68K daltons. Amino acid analyses indicated a subunit molecular weight of 68,042. Molecular sieve chromatography in the presence of 10% (NH4)2SO4 showed that the molecular weight of the native enzyme was 127K daltons. These values indicate that the native enzyme is composed of two identical subunits. However, the purified enzyme gave 4 protein bands on polyacrylamide gel electrophoresis under non-denaturing conditions, and appeared as a single fraction in the vicinity of the void volume on Ultrogel AcA 34 column chromatography at low salt concentration, indicating that its molecular weight exceeded 200K daltons. These findings indicate that the enzyme tends to aggregate owing to its own physicochemical characteristics. The Km values for IMP and NAD were calculated to be 12 and 25 microM, respectively, and the Ki values for XMP, GMP, and AMP to be 109, 130, and 854 microM, respectively. The purified enzyme showed full activity in the presence of K+, and K+ could be partially replaced by Na+. PCMB inactivated the enzyme, but the activity was completely restored by the addition of DTT. Cl-IMP also inactivated the enzyme and IMP prevented this inactivation.(ABSTRACT TRUNCATED AT 250 WORDS)

Inosine 5'-monophosphate dehydrogenase of Escherichia coli. Purification by affinity chromatography, subunit structure and inhibition by guanosine 5'-monophosphate.

Escherichia coli IMP dehydrogenase (EC 1.2.1.14) was purified by affinity chromatography on immobilized nucleotides. The enzyme binds to agarose-bound 8-(6-aminohexyl)-AMP, N6-(6-aminohexyl)-AMP and 8-(8-amino-octyl)-IMP but not to immobilized NAD+ or Cibacron Blue F3G-A. AMP proved to be an effective eluent. A large-scale purification scheme in which 8-(6-aminohexyl)-AMP-agarose was used resulted in a homogeneous preparation of IMP dehydrogenase. The enzyme was also purified by immunoprecipitation with monospecific antisera. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, N-terminal amino acid analysis and tryptic 'finger-printing' demonstrated that IMP dehydrogenase comprises identical subunits of mol.wt. 58000. Trypsin and Pronase cleave the 58000-mol.wt. subunit into peptides of mol.wts. 42000 and 14000, with a concomitant decrease in enzyme activity. These observations rationalize much of the contradictory data on the subunit composition of the enzyme found in the literature. GMP appears to be a competitive inhibitor with respect to IMP, with no evidence for regulatory behaviour being found. The two purification procedures were also used to purify inactive mutant enzymes from guaB mutant strains of E. coli.