Dynamic compartmentalization of purine nucleotide metabolic enzymes at leading edge in highly motile renal cell carcinoma.

Compartmentalization is vital for biological systems at multiple levels, including biochemical reactions in metabolism. Organelle-based compartments such as mitochondria and peroxisomes sequester the responsible enzymes and increase the efficiency of metabolism while simultaneously protecting the cell from dangerous intermediates, such as radical oxygen species. Recent studies show intracellular nucleotides, such as ATP and GTP, are heterogeneously distributed in cells with high concentrations at the lamellipodial and filopodial projections, or leading edge. However, the intracellular distribution of purine nucleotide enzymes remains unclear. Here, we report the enhanced localization of GTP-biosynthetic enzymes, including inosine monophosphate dehydrogenase (IMPDH isotype 1 and 2), GMP synthase (GMPS), guanylate kinase (GUK1) and nucleoside diphosphate kinase-A (NDPK-A) at the leading edge in renal cell carcinoma cells. They show significant co-localization at the membrane subdomain, and their co-localization pattern at the membrane is distinct from that of the cell body. While other purine nucleotide biosynthetic enzymes also show significant localization at the leading edge, their co-localization pattern with IMPDH is divergent. In contrast, a key glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), predominantly localized in the cytoplasm. Mechanistically, we found that plasma membrane localization of IMPDH isozymes requires active actin polymerization. Our results demonstrate the formation of a discrete metabolic compartment for localized purine biosynthesis at the leading edge, which may promote localized nucleotide metabolism for cell migration and metastasis in cancers.

Determination of inosine 5'-monophosphate dehydrogenase activity in red blood cells of thiopurine-treated patients using HPLC.

Thiopurine drugs are commonly used in immune diseases and to a lesser extent, in transplant rejection prophylaxis: however interindividual variability in drug response and in the occurrence of adverse events is observed. Genetic variation in thiopurine S-methyltransferase (TPMT) doesn't completely explain the occurrence of all adverse events and drug response variability. The potential implication of other enzymes involved in thiopurine metabolism, such as ITPA, has been investigated over the last decade but little data is available on inosine 5'-monophosphate dehydrogenase (IMPDH) in patients treated with thiopurine drugs. The authors reported a HPLC method to determine IMPDH activity in the red blood cells (RBCs) of thiopurine-treated patients. IMPDH activity was evaluated by enzymatic conversion of inosine 5'-monophosphate (IMP) to xanthosine 5'-monophosphate (XMP). The XMP formed was analyzed on a Luna® NH2 stationary phase, a weak anion exchange phase that exhibits both ionic and hydrophobic properties. XMP was eluted below 15min. Intra-assay and inter-assay precisions were below 9% for RBCs supplemented with 2, 40 and 80µmol/L of XMP. IMPDH activity was measured in adults without thiopurine treatment as well as in adult and paediatric patients treated with thiopurines. A wide interindividual variability in IMPDH activity in RBCs was observed. No difference in IMPDH activity was found between untreated subjects and adult and paediatric patients on thiopurine therapy (median value 11.8, 7.9 and 7.7nmol XPM/g Hb/h respectively). The method described is useful in the determination of IMPDH phenotype from patients on thiopurine therapy and in the investigation of the potential relationship between IMPDH activity in RBCs and the occurrence of adverse events and drug response variability.

Quantitative analysis of purine nucleotides indicates that purinosomes increase de novo purine biosynthesis.

Enzymes in the de novo purine biosynthesis pathway are recruited to form a dynamic metabolic complex referred to as the purinosome. Previous studies have demonstrated that purinosome assembly responds to purine levels in culture medium. Purine-depleted medium or 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT) treatment stimulates the purinosome assembly in HeLa cells. Here, several metabolomic technologies were applied to quantify the static cellular levels of purine nucleotides and measure the de novo biosynthesis rate of IMP, AMP, and GMP. Direct comparison of purinosome-rich cells (cultured in purine-depleted medium) and normal cells showed a 3-fold increase in IMP concentration in purinosome-rich cells and similar levels of AMP, GMP, and ratios of AMP/GMP and ATP/ADP for both. In addition, a higher level of IMP was also observed in HeLa cells treated with DMAT. Furthermore, increases in the de novo IMP/AMP/GMP biosynthetic flux rate under purine-depleted condition were observed. The synthetic enzymes, adenylosuccinate synthase (ADSS) and inosine monophosphate dehydrogenase (IMPDH), downstream of IMP were also shown to be part of the purinosome. Collectively, these results provide further evidence that purinosome assembly is directly related to activated de novo purine biosynthesis, consistent with the functionality of the purinosome.

Elevated expression of IMPDH2 is associated with progression of kidney and bladder cancer.

Novel molecular markers for cancer progression are valuable for the diagnosis and evaluation of treatment efficacies of the diseases. Expression of inosine 5'-monophosphate dehydrogenase type II (IMPDH2), a rate-limiting enzyme in the de novo guanine nucleotide biosynthesis, is up-regulated in various neoplasms, including prostate cancer and patient serum. However, whether IMPDH2 can serve as a biomarker for other urologic cancers is unknown. Paired patient tissue macroarrays were analyzed by immunohistochemistry, the IMPDH2 protein expression in these tissues was quantitated and expressed as immunoreactivity scores. Compared with non-cancerous tissues, IMPDH2 protein expression levels were significantly upregulated in kidney and bladder cancer, but no difference in testis cancer. In addition, expression of IMPDH2 was not associated with the disease clinical stages and pathological features. The findings suggest that overexpressed IMPDH2 can be used as a biomarker for kidney and bladder cancer diagnosis and is a potential therapeutic target for the diseases.

Inosine 5'-monophosphate dehydrogenase activity as a biomarker in the field of transplantation.

Inosine 5'monophosphate dehydrogenase (IMPDH) is the rate limiting enzyme in the de novo synthesis of guanine nucleotides. The direct determination of target enzyme activity as a biomarker of mycophenolic acid (MPA) may help to estimate better the individual response to the immunosuppressant. However, the assessment of the clinical utility of this approach is limited by the diversity of the assay systems, which has not yet allowed the prospective assessment of this enzyme in larger patient cohorts. A recently validated and standardized assay allows the investigation of IMPDH activity in larger clinical studies. Although descriptive results from observational studies hold promise for a more individualized therapy in transplant medicine, more studies are needed to prospectively validate this approach.

Proteomic analysis reveals metabolic changes during yeast to hypha transition in Yarrowia lipolytica.

Fungal dimorphism is important for survival in different environments and has been related to virulence. The ascomycete Yarrowia lipolytica can grow as yeast, pseudomycelial or mycelial forms. We have used a Y. lipolytica parental strain and a Deltahoy1 mutant, which is unable to form hypha, to set up a model for dimorphism and to characterize in more depth the yeast to hypha transition by proteomic techniques. A two-dimensional gel electrophoresis (2-DE) based differential expression analysis of Y. lipolytica yeast and hyphal cells was performed, and 45 differentially expressed proteins were detected; nine with decreased expression in hyphal cells were identified. They corresponded to the S. cerevisiae homologues of Imd4p, Pdx3p, Cdc19, Sse1p, Sol3p, Sod2p, Xpt1p, Mdh1p and to the unknown protein YALIOB00924g. Remarkably, most of these proteins are involved in metabolic pathways, with four showing oxidoreductase activity. Furthermore, taking into account that this is the first report of 2-DE analysis of Y. lipolytica protein extracts, 35 more proteins from the 2D map of soluble yeast proteins, which were involved in metabolism, cell rescue, energy and protein synthesis, were identified.

HPLC/tandem ion trap mass detector methods for determination of inosine monophosphate dehydrogenase (IMPDH) and thiopurine methyltransferase (TPMT).

The efficiency of Mycophenolate mofetil (MMF) and Azathioprine (AZA) as immunosuppressive agents depends on the activity of 2 enzymes, inosine monophosphate dehydrogenase (IMPDH) and thiopurine methyltransferase (TPMT) respectively. We present preliminary evaluation of nonradioactive methods that apply HPLC with ion-trap mass detection to measure the activities of IMPDH in peripheral blood mononuclear cells (PBMC) and TPMT in the erythrocytes (RBC). We found IMPDH activity of 0.9 +/- 0.2 nmol/hour/10(6) PBMC and TPMT activity of 19.9 +/- 4.7 nmol/hour/ml RBC in healthy subjects. These methods, following its further validation, could be useful for monitoring the activity in a clinical and experimental setting.

Inosine 5'-monophosphate dehydrogenase binds nucleic acids in vitro and in vivo.

Inosine 5'-monophosphate dehydrogenase (IMPDH) is the rate-limiting enzyme in the de novo biosynthesis of guanine nucleotides. In addition to the catalytic domain, IMPDH contains a subdomain of unknown function composed of two cystathione beta-synthase domains. Our results, using three different assays, show that IMPDHs from Tritrichomonas foetus, Escherichia coli, and both human isoforms bind single-stranded nucleic acids with nanomolar affinity via the subdomain. Approx. 100 nucleotides are bound per IMPDH tetramer. Deletion of the subdomain decreases affinity 10-fold and decreases site size to 60 nucleotides, whereas substitution of conserved Arg/Lys residues in the subdomain with Glu decreases affinity by 20-fold. IMPDH is found in the nucleus of human cells, as might be expected for a nucleic-acid-binding protein. Lastly, immunoprecipitation experiments show that IMPDH binds both RNA and DNA in vivo. These experiments indicate that IMPDH has a previously unappreciated role in replication, transcription or translation that is mediated by the subdomain.

Aptamer-based biosensor arrays for detection and quantification of biological macromolecules.

We have developed a chip-based biosensor for multiplex analysis of protein analytes. The biosensor utilizes immobilized DNA and RNA aptamers, selected against several different protein targets, to simultaneously detect and quantify levels of individual proteins in complex biological mixtures. Aptamers were each fluorescently labeled and immobilized on a glass substrate. Fluorescence polarization anisotropy was used for solid- and solution-phase measurements of target protein binding. We show that solid-phase aptamer-protein interactions recapitulate binding interactions seen in solution. Furthermore, we demonstrate specific detection and quantitation of cancer-associated proteins (inosine monophosphate dehydrogenase II, vascular endothelial factor, basic fibroblast growth factor) in the context of human serum and in cellular extracts. It is expected that this technology could speed diagnosis of cancer by enabling direct detection of the expression and modification of proteins closely correlated with disease.

Non-radioactive determination of inosine 5'-monophosphate dehydro-genase (IMPDH) in peripheral mononuclear cells.

BACKGROUND: The immunosuppressive activity of mycophenolate mofetil (MMF) is based on the reversible inhibition of inosine 5'-monophosphate dehydrogenase (IMPDH) by mycophenolic acid (MPA). It was the aim of this study to develop a nonradioactive method for specific measurement of IMPDH activity in isolated peripheral mononuclear cells (MNC). METHODS: The procedure is based on the incubation of lysed MNC with inosine 5'-monophosphate (IMP) followed by direct chromatographic determination of produced xanthosine 5'-monophosphate (XMP). IMPDH activity was measured in MNC of MMF-treated patients and nontreated volunteers. RESULTS: The within-run (n = 10) and between-run (n = 20) coefficients of variation (CV) for IMPDH activity were < 8% and < 10%, respectively. IMPDH activity in 60 healthy volunteers (19-63 yr) ranged from 4.72 to 32.92 nmol/h/mg protein (mean = 18.39 +/- 6.24). The IC(50) for in vitro inhibition of IMPDH activity was about 2 to 3 microg/L. Application of a single dose of 1 g MMF in dialysis patients resulted in a significant inhibition (by 47-95%; p < 0.05) of IMPDH activity in lysed MNC. CONCLUSIONS: The proposed assay specifically and reliably measures IMPDH activity in MNC. The procedure is applicable to evaluate pharmacodynamic activity in MMF-treated patients. The observed interindividual variability of IMPDH activity may reflect pharmacodynamic differences in MMF-treated patients.

Pharmacodynamic monitoring of immunosuppressive drugs.

Pharmacodynamic (PD) monitoring measures the biological response to a drug, which alone--or coupled with pharmacokinetics--provides a novel method for optimization of drug dosing. PD monitoring has been investigated by us and other investigators primarily for four immunosuppressive drugs: cyclosporine (CsA), azathioprine (AZA), mycophenolate mofetil (MMF), and rapamycin (RAPA). PD monitoring of CsA and MMF involves measuring the activity of the enzymes calcineurin and inosine monophosphate dehydrogenase, respectively. The PD of AZA is assessed by measuring the activity of thiopurine methyltransferase, which is induced by a metabolite of AZA, 6-mercaptopurine. The PD for RAPA involves measuring the activity of a P70 S6 kinase in lymphocytes. To date, the most detailed studies have been performed with PD monitoring of CsA and MMF. Similarities exist in the PD responses to CsA and MMF in renal-transplant patients. At trough concentrations in blood, both drugs reduce the activity of their target enzymes by only 50%; however, considerable interpatient variability is evident. Throughout the dosing interval, the enzyme activities parallel the respective drug concentrations. AZA treatment of renal-transplant patients who exhibited an increase in thiopurine methyltransferase activity from time of transplantation resulted in fewer episodes of active rejection. Additional clinical trials are currently underway to relate various pharmacokinetics and PD parameters to clinical response, to ascertain which provides the best guide for dosing. PD monitoring may provide an alternative approach to additional measurements of drug concentrations.

A two-dimensional electrophoresis database of human breast epithelial cell proteins.

As sequencing of the human genome progresses, attention is turning to when and where specific genes are being expressed and how that expression is regulated. The human breast, with the highly specific, but transient, function of milk production (lactation), exemplifies human gene regulation. The molecular mechanisms for the dramatic structural and functional changes involved in shifting from lactation-capable to lactation-incapable tissue are poorly understood, as are the mechanisms that result in deviation from normal breast cell growth into different types of breast neoplasms. We are using quantitative two-dimensional electrophoresis (2-DE) to determine which proteins are present in different types of human breast cells (milk-producing and -nonproducing, estrogen-receptor-positive and -negative, normal and malignant) and which proteins change in abundance in response to stimuli that trigger cell differentiation, growth, or death. A composite map of proteins found in human breast cells is being generated and used as an index of human genes that are differentially expressed, both qualitatively and quantitatively. Proteins found in 15 different types of human breast cells, two from healthy tissue (from milk and reduction mammoplasty tissue) and 13 from tumor tissue, are now included in the composite map. Copies of the human breast epithelial cell protein map are available on the World Wide Web (URL: http:(/)/www.anl.gov/CMB/PMG/ projects/index_hbreast.html) with links to quantitative data and identifications for proteins found to be differentially expressed in these epithelial cells. Links to the Swiss-Prot and enzyme metabolic pathway databases are also provided. The World Wide Web presentation is designed to allow public access to the available 2-DE data together with logical connections to databases providing genome-related information.

[An enzyme study of purine nucleotide biosynthesis in the plerocercoids of cestodes in the fam. Ligulidae]. / Izuchenie fermentov biosinteza purinovykh nukleotidov u plevrotserkoidov tsestod sem. Ligulidae.

By means of spectrophotometric method there was determined the activity of three enzymes of biosynthesis of purine nucleotides: amino imidazole ribonucleotide-carboxylase (AIR-carboxylase, EC 4.1.1.21), an enzyme of biosynthesis of purine nucleotides de novo in plerocercoids of Schistocephalus pungitii and Digramma interrupta; inosine monophosphate-dehydrogenase (IMPh-dehydrogenase, EC 1.2.1.14), an enzyme of salvage path, and adenylosuccinate lyase (EC 4.3.2.2), an enzyme taking part both in biosynthesis de novo and salvage in plerocercoids of Schistocephalus pungitii. The activity of AIR-carboxylase was not determined. Specific activities of adenylosuccinate lyase and IMPh-dehydrogenase amount to (1.3 +/- 0.3) x 10(-3) and (1.2 +/- 0.4) x 10(-3) mumole/min.mg protein, respectively. The activity of the three enzymes was determined in the liver of ten-spined stickleback, a host of S. pungitii plerocercoids. The question of metabolic dependence of Ligulidae plerocercoids on hosts to provide for purine bases is discussed.

Regulation of IMP dehydrogenase gene expression by its end products, guanine nucleotides.

To study the regulation of IMP dehydrogenase (IMPDH), the rate-limiting enzyme of guanine nucleotide biosynthesis, we examined the effects of nucleosides, nucleotides, nucleotide analogs, or the IMPDH inhibitor mycophenolic acid (MPA) on the steady-state levels of IMPDH mRNA. The results indicated that IMPDH gene expression is regulated inversely by the intracellular level of guanine ribonucleotides. We have shown that treatment with guanosine increased the level of cellular guanine ribonucleotides and subsequently reduced IMPDH steady-state mRNA levels in a time- and dose-dependent manner. Conversely, MPA treatment diminished the level of guanine ribonucleotides and increased IMPDH mRNA levels. Both of these effects on the steady-state level of IMPDH mRNA could be negated by cotreatment with guanosine and MPA. The down regulation of IMPDH gene expression by guanosine or its up regulation by MPA was not due to major changes in transcriptional initiation and elongation or mRNA stability in the cytoplasm but rather was due to alterations in the levels of the IMPDH mRNA in the nucleus. These results suggest that IMPDH gene expression is regulated by a posttranscriptional, nuclear event in response to fluctuations in the intracellular level of guanine ribonucleotides.

Induction of cell differentiation in melanoma cells by inhibitors of IMP dehydrogenase: altered patterns of IMP dehydrogenase expression and activity.

To study the induction of differentiation in human melanoma cells, we treated 12 melanoma cell lines with mycophenolic acid and tiazofurin, inhibitors of IMP dehydrogenase (IMPDH). In all cell lines studied, both agents inhibited cell growth and increased melanin content. However, the degree of growth inhibition did not necessarily correspond to the increase in melanin content. A detailed analysis of the HO and SK-MEL-131 cell lines indicated that mycophenolic acid and tiazofurin caused a time- and dose-dependent increase in the expression of a series of other maturation markers, including formation of dendrite-like structures, tyrosinase activity, and reactivity with the CF21 monoclonal antibody. The growth inhibition and melanogenesis induced by the IMPDH inhibitors was abrogated by the addition of exogenous guanosine. No such effect was observed after treatment of the cells with phorbol 12-myristate 13-acetate or retinoic acid, two other inducers of differentiation in these cells. The mycophenolic acid- and tiazofurin-treated cells also showed an increased level of IMPDH mRNA and protein, perhaps because of compensation for the inhibitor-mediated decrease in IMPDH activity. In contrast, treatment with phorbol 12-myristate 13-acetate or retinoic acid resulted in decreased levels of IMPDH mRNA and protein. The lack of a consistent pattern of IMPDH expression in the cells treated with IMPDH inhibitors and phorbol 12-myristate 13-acetate or retinoic acid suggests that the altered expression of IMPDH is not a general requirement for the induction of cell differentiation in these cells. Our results also suggest that IMPDH inhibitors may provide a useful approach to circumvent the differentiation block in melanoma.

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.

Inosine monophosphate dehydrogenase activity in acute leukaemia.

Inosine monophosphate dehydrogenase (IMPD) is an important enzyme in de-novo purine synthesis. The level of IMPD activity has been suggested to determine whether acute leukaemia cells proliferate (if the activity is high) or differentiate (if IMPD activity is low). IMPD activity measured by the conversion of inosine monophosphate to xanthine monophosphate ranged from 12.5 to 87.0 (mean 49.4) pmol/h/10(6) cells in normal bone marrow. The levels were significantly raised in AML (range 14-374, mean 184 pmol/h/10(6) cells) and ALL (range 65-228, mean 172 pmol/h/10(6) cells). Normal tonsillar (B) lymphocytes showed higher levels (range 78-159, mean 110 pmol/h/10(6) cells) than resting peripheral blood T lymphocytes (range 8.8-51.2, mean 28.1 pmol/h/10(6) cells). In CLL, the results (range 19-173, mean 64.3 pmol/h/10(6) cells) were comparable to those of normal tonsillar B lymphocytes. IMPD levels could be related to cell cycle in PHA-stimulated lymphocytes, since IMPD activity increased in parallel with increase in DNA synthesis measured by labelled thymidine incorporation. On the other hand, IMPD activity did not correlate with the proportion of proliferating cells measured on a FACS sorter in either AML or ALL or in normal tonsillar B cells. We conclude that IMPD levels are higher in B than T lymphocytes and in acute leukaemia blasts compared to more differentiated mixed bone marrow cells. The results do not suggest, however, that IMPD assay will be of value in differentiation of the various subtypes of acute leukaemia or of malignant haemopoietic cells from the equivalent normal cell at the same level of differentiation.

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).