Metabolism of Purines and Pyrimidines in Health and Disease

39th Meeting of the Polish Biochemical Society Gdañsk 16–20 September 2003

SESSION 6

Metabolism of purines and pyrimidines in health and disease

Organized by A. C. Sk³adanowski, A. Guranowski 182 Session 6. Metabolism of purines and pyrimidines in health and disease 2003

323 Lecture The role of DNA methylation in cytotoxicity mechanism of adenosine analogues in treatment of leukemia Krystyna Fabianowska-Majewska Zak³ad Chemii Medycznej IFiB, Uniwersytet Medyczny, ul. Mazowiecka 6/8, 92 215 £ódŸ

Changes in DNA methylation have been recognized tory effects of cladribine and fludarabine on DNA as one of the most common molecular alterations in hu- methylation, after 48 hr growth of K562 cells with the man neoplastic diseases and hypermethylation of drugs, are non-random and affect mainly CpG rich is- gene-promoter regions is one of the most frequent lands or CCGG sequences but do not affect sepa- mechanisms of the loss of gene functions. For this rea- rately-located CpG sequences. The analysis showed son, DNA methylation may be a tool for detection of that cladribine (0.1 mM) reduced the methylated early cell transformations as well as predisposition to cytosines in CpG islands and CCGG sequences to a sim- metastasis process. Moreover, DNA methylation seems ilar degree. The inhibition of cytosine methylation by to be a promissing target for new preventive and thera- fludarabine (3 mM) was observed mainly in CCGG se- peutic strategies. quences, sensitive to HpaII, but the decline in the meth- Our studies on DNA methylation and cytotoxicity ylated cytosine, located in CpG island was 2-fold lower mechanism of antileukemic drugs, cladribine and than that with cladribine. In contrast, the action of fludarabine (adenosine analogues), indicate that the decitabine (a commonly used potent inhibitor of DNA drugs lead to significant decrease of DNA methyltransferase activity) is non-specific to cytosine methyltransferase activity in K562, L1210 and human localization, because it causes a decrease of methylated stimulated T lymphocytes. Moreover, in our studies cytosines mainly in isolated CpG sequences and, to a with exogenic bacterial methylase enzyme, we observed lesser degree, in both CCGG sequences and CpG is- a significant decrease of DNA methylation level after lands. the growth of K562 and L1210 cells in the presence of The effects of both of the antileukemic drugs, leading cladribine and fludarabine. DNA methylation-de- to a substantial drop in methylated cytosine in CpG is- pendent restriction analysis with HpaII and BssHII en- lands may be significant in the therapy of cancers asso- zymes which recognize unmethylated CCGG and ciated with gene silencing due to hypermethylation of GCGCGC sequences, respectively, showed that inhibi- their regulatory regions.

324 Lecture Current knowledge about the metabolism of mono- and di-nucleoside polyphosphates in plants Andrzej Guranowski Department of Biochemistry and Biotechnology, University of Agriculture, ul. Wo³yñska 35, 60-637 Poznañ

Mononucleoside polyphosphates (pnN), such as zymes occurring in plants such as phosphatase and adenosine 5’-tetraphosphate (p4A) and adenosine 5’-pen- apyrase [4], that can hydrolyze p4A or p5A, and taphosphate (p5A), and dinucleoside polyphosphates phosphodiesterase I, for which Ap3A and Ap4A are very (NpnN’), such as diadenosine 5’,5’’’-P1,P3-triphosphate good substrates [1]. (Ap3A) or diadenosine 5’,5’’’-P1,P4-tetraphosphate Several enzymes forming an acyl-adenylate interme- (Ap4A), presumably occur in all organisms, however, diate with concomitant release of PPi can catalyze the their presence in plants has not yet been demonstrated. synthesis of different pnNs and NpnN’s. Phenylalanyl- The existence of highly specific enzymes that catalyze the and seryl-tRNA synthetases from yellow lupin seeds [5] hydrolysis of (di)nucleoside polyphosphates, such as and recently described 4-coumarate:coenzyme A ligase (asymmetrical) dinucleoside tetraphosphatase (EC from Arabidopsis thaliana [6] are so far the only plant 3.6.1.17) [1], dinucleoside triphosphatase (EC 3.6.1.29) enzymes known to synthesize NpnN’s and/or pnNs. Ki- [1, 2] and nucleoside 5’-tetraphosphatase (EC 3.6.1.14) netic and molecular properties of enzymes involved in [3], indicates that these uncommon nucleotides may exist the metabolism of pnNs and NpnN’s in plants will be and have a function in planta. In addition, these com- presented and biological role of these compounds dis- pounds can be substrates for nonspecific catabolic en- cussed. 2003 39th Meeting of the Polish Biochemical Society 183

1. Jakubowski H, Guranowski A (1983) J Biol Chem, 258: 4. Guranowski A, Starzyñska E, Rataj-Guranowska M, 9982–9989. Günther Sillero MA (1991) Protein Expression Purif, 2: 2. Guranowski A, Starzyñska E, Bojarska E, Stêpiñski J, 235–239. Dar¿ynkiewicz E (1996) Protein Expression Purif, 8: 5. Jakubowski H (1983) Acta Biochim Polon, 30: 51–69. 416–422. 6. Pietrowska-Borek M, Stuible H-P, Kombrink E, Guranowski 3. Guranowski A, Starzyñska E, Brown, P, Blackburn GM A (2003) Plant Physiol, 131: 1401–1410. (1997) Biochem J, 328: 257–262.

325 Lecture Flaviviridae viruses NTPase/helicase: a new target for inhibitors Tadeusz Kulikowski Instytut Biochemii i Biofizyki, Polska Akademia Nauk, ul. Pawiñskiego 5A, 02-106 Warszawa

The Flaviviridae family include small viruses, which volved in transcription and replication of viral RNA sin- genom consists of a single-stranded positive-sense gle-stranded genomes. The crucial role of the enzyme RNA. Among them are dangerous human and animal for the virus replication cycle was demonstrated in ex- pathogenic viruses such as hepatitis C virus (HCV), periments using NTPase/helicase specific inhibitors. West Nile virus (WNV), and viral encephalitis (JEV). This make the enzyme an attractive target for develop- The genome of these viruses encodes 3 structural and ment of Flaviviridae-specific antiviral therapies. The 7 non-structural proteins. The enzymatic activities of present study involves investigation of the broad spec- NTPase/helicase were detected in the carboxy-terminal trum of activators and inhibitors of the enzymatic activ- fragment of the non-structural protein 3 (NS3) of the vi- ities of Flaviviridae NTPase/ helicase and describes the ruses. RNA nucleoside triphosphatase (NTPase)/ different mechanisms by which the inhibitors could act. helicases represent a large family of proteins that are Some of them, especially some modified benzimi- ubiquitously distributed over a wide range of organ- dazoles and benzotriazoles, show potential utility as an- isms. These enzymes play essential role in cell develop- tiviral agents against Flaviviridae viruses. ment and differentiation, and some of them are in-

326 Lecture The role of insulin in adenosine metabolism and action Tadeusz Pawe³czyk Zak³ad Medycyny Molekularnej, Akademia Medyczna w Gdañsku, ul. Dêbinki 7, 80-211 Gdañsk

Adenosine plays an important role in physiology of Adenosine exerts its physiological effect by coupling several organs. Once generated, adenosine could be to cell-surface receptors (A1, A2a, A2b, A3). It has been deaminated to inosine by adenosine deaminase, reported that the responsiveness of various tissues to phosphorylated to AMP by adenosine kinase (AK), or adenosine in diabetes is altered. Investigation of rat di- transported into extracellular fluid. Extracellular me- abetic tissues revealed that mRNA level of A1 receptor tabolism of nucleotides produces adenosine, which is was significantly increased in diabetic kidney and was taken up by the cell or deaminated to inosine. Under unchanged in diabetic heart and liver. The expression normal conditions most of the adenosine formed in the of A2b receptor was decreased in diabetic kidney and cell is phosphorylated to AMP by adenosine kinase. Re- was unchanged in diabetic heart and liver. The level of cently reported data indicate that the activity and A2a mRNA was decreased in diabetic liver, whereas in mRNA level of adenosine kinase is significantly low- diabetic heart and kidney expression of this receptor ered in diabetic rats suggesting that the expression was unchanged. Administration of insulin to diabetic level of AK gene is dependent on insulin. Further exper- rats restored normal level of mRNA for adenosine re- iments performed on cultured rat lymphocytes have ceptors. These data suggest that insulin may affect the demonstrated that insulin induces AK gene expression expression level of adenosine receptors in a tissue spe- in a dose- and time-dependent manner. It has been dem- cific manner. onstrated that insulin signal to AK gene in rat lympho- The affinity for adenosine varies between receptors, cytes is transmitted by the MAP kinases pathway. thus its activation depends on adenosine concentra- 184 Session 6. Metabolism of purines and pyrimidines in health and disease 2003 tion. On the other hand, the level of adenosine depends insulin. Whereas, decreased expression of ENT1 re- on its metabolism and transport across plasma mem- sulted from increased glucose concentration but not branes. Thus, carrier-mediated transport of adenosine from changes in insulin level. is likely to play an important role in modulating cell In conclusion, presented data indicate that insulin function, because efficiency of the transport processes has profound effect on adenosine metabolism, trans- may determine adenosine availability either to recep- port and receptors. Therefore, the above-mentioned al- tors or to metabolizing enzymes. In diabetic rats the ex- terations in adenosine metabolism and handling may pression level of nucleoside transporters is altered in a play an important role in development of many diabetic tissue specific manner. Administration of insulin to di- complications. abetic rats resulted in normalization of nucleoside References: transporters expression level except nitrobenzyl- 1. Pawe³czyk T, Sakowicz M, Szczepañska-Konkel M, thioinosine (NBMPR)-sensitive equilibrative trans- Angielski S (2000) Arch Biochem Biophys, 375: 1–6. porter (ENT1), which remained lowered. Experiments 2. Sakowicz M, Pawe³czyk T (2002) Mol Cell Biochem, 236: 163–171. on cultured rat cardiac fibroblasts and rat lymphocytes + 3. Pawe³czyk T, Sakowicz M, Podgórska M, Szcze- have showed that expression of Na -dependent pañska-Konkel M (2003) Exp Cell Res, 281: 152–163. nucleoside transporters (CNT1, CNT2) and equili- 4. Pawe³czyk T, Podgórska M, Sakowicz M (2003) Mol brative NBMPR-insensitive transporter is regulated by Pharmacol, 60: 81–88.

327 Lecture Thymidylate biosynthesis as a target in chemotherapy. Potential importance of a peculiar expression pattern of enzymes involved in thymidylate biosynthesis in nematodes Wojciech Rode, Magdalena D¹browska, Barbara Go³os, Patrycja Wiñska, El¿bieta Jagielska, Joanna Cieœla, Zbigniew Zieliñski Nencki Institute of Experimental Biology, Nencki Institute of Experimental Biology, ul. Pasteura 3, 02-093 Warszawa

Thymidylate (dTMP), is formed in cells de novo in a rested: muscle larvae of both Trichinella species and C. process of the C(5) methylation of 2’-deoxyuridylate elegans dauer larvae. TS mRNA was also persistently (dUMP), catalyzed by thymidylate synthase (TS) and expressed throughout T. spiralis development. By con- involving a concerted transfer and reduction of the focal microscopy the enzyme protein was shown to be one-carbon group of N5,10-methylenetetrahydrofolate, present in apparently all muscle larva cells. Of particu- with concomitant production of dTMP and dihydro- lar interest is the presence of high activities of TS and folate. The latter is reduced by dihydrofolate reductase dUTPase, as neither proliferation nor DNA biosyn- (DHFR) to regenerate tetrahydrofolate. One of the thesis may be expected to occur to any significant ex- sources of TS substrate, dUMP, is hydrolysis of the cor- tent at those developmental stages. As TS induction responding triphosphate, dUTP, in a pyrophosphatase has been shown to be due to leaving quiescence (cell cy- reaction catalyzed by dUTPase. TS and dUTPase induc- cle G0 phase activity), high enzyme level observed in tion is known to be associated with cell proliferation. the developmentally arrested larvae of different nema- Inhibition of dTMP synthesis by drugs targeted at ei- todes may be due to their cells being arrested in the cell ther TS (5-fluorouracil, raltitrexed), or DHFR (metho- cycle, pointing to some cell cycle regulation peculiarity, trexate, trimetrexate) is taken advantage of in chemo- apparently common for parasitic and free-living nema- therapy. todes. Such a peculiarity would be obviously interesting Prompted by our earlier finding of high specific activi- as a potential chemotherapeutic target. In view of the ties of TS, DHFR and dUTPase in crude extracts of latter, high TS expression is of interest, as the enzyme non-developing muscle larvae of T. spiralis, we studied has been ascribed a regulatory (in addition to the cata- developmental patterns of the same enzyme activities lytic) function, based on its apparent ability to bind sev- in parasitic (T. spiralis and T. pseudospiralis) and eral mRNA species, thus regulating translation of the free-living (Caenorhabditis elegans) nematodes, and corresponding proteins, between them c-myc and p53 found them persistently expressed at all developmental involved in cell cycle regulation. stages studied, including those developmentally ar- Supported by the State Committee for Scientiic Research (KBN, Poland) grant 6PO4C 003 21 2003 39th Meeting of the Polish Biochemical Society 185

328 Lecture Regulation and function of ecto-5’-nucleotidase and adenosine in cancer Jozef Spychala Lineberger Comprehensive Cancer Center, University of North Carolina, Mason Farm Rd., 27599-7295 Chapel Hill, USA

Adenosine has potent angiogenic, cytoprotective and pressed in cells that lack eN. Remarkably, the expres- anti-inflammatory functions that have been docu- sion profile of membrane, cytoskeletal and signaling mented in several tissues. Increasing evidence suggests molecules is almost identical in WI38 fibroblast sug- that similar functions of adenosine may contribute to gesting that transition to ER(–) phenotype follows epi- solid tumor progression. Since in epithelial cells thelial to mesenchymal transdifferentiation (EMT). adenosine is generated by ecto-5’-nucleotidase (eN), we Analysis of eN expression in clinical breast cancer sam- focused our analysis on the expression and regulation ples confirmed negative correlation with ER receptor of eN in cancer. We found that eN is upregulated by status. In addition, preliminary assesment of eN ex- several oncogenes, including c-jun, b-catenin and wnt. pression in ER(–) breast cancer samples showed high In breast carcinoma eN expression is suppressed by eN expression at the invasive end of cancer lesions and ERa and loss of estrogen receptor is prerequisite for co-expression with vimentin and integrin b1. The in- high eN expression. Expression profiling of breast can- creased expression of eN in cancer cells was also associ- cer cells revealed that eN expression is tightly associated with altered adhesive and migratory properties ated with metastatic phenotype that is more typical for and we hypothesize that either higher level of produced ER(–) cells. Among markers characteristic for invasive adenosine by eN or interaction of this membrane pro- cancer cells eN co-expresses with CD44, EGFR, MDR1, tein with the ECM and modulation of integrin function Integrins a5b1, and a3b1 and vimentin. Other cell sur- may contribute to more aggressive behaviour of ER(-) face antigens previously established as markers of and metastatic cancer cells. breast crcinoma, such as CD24 and uPAR, were ex-

329 Lecture Role of P1 and P2 purinoceptors in the regulation of glomerular filtration rate Miros³awa Szczepañska-Konkel Katedra i Zak³ad Analityki Klinicznej, Akademia Medyczna w Gdañsku, ul. Dêbinki 7, 80-041 Gdañsk

Extracellular dinucleotides (ApnA) and their metabo- tion of P2x, ligand-gated ion channels receptors. We lites including ATP, ADP, and adenosine have been rec- have also shown that the effects of adenosine on ognized as modulators of renal vascular tone and in intracapillary volume of glomeruli are mediated pre- consequence renal hemodynamics. Their effects are dominantly by action on P1-receptors of the A1 and brought about by activation of membrane bound P1- A2a — subtypes. Adenosine induces the contraction of and P2- purinoceptors located on smooth muscle and glomeruli via A1 receptors (negatively coupled to endothelial cells. We have indicated that adenosine, adenylate cyclase), and this effect is limited by activity ATP, Ap3A, Ap4A, Ap5A (at nanomolar concentration) of A2a receptors (positively coupled to adenylate act on glomerular microvasculature through activation cyclase). Final effect of adenosine action on intra- of metabotropic (P1, P2Y) and ionotropic receptors capillary volume of glomeruli depends on relation be- (P2X) located on glomerular cells (mesangial, endothe- tween A1 and A2 receptors which may change in vari- lial and epithelial cells). These compounds change the ous (patho)physiological conditions. For example, re- intracapillary volume of isolated glomeruli by mediat- nal glomeruli of insulin dependent diabetes mellitus ing either direct constrictor effects on mesangium or rats with hyperfiltration show an attenuated respon- relaxant effects through stimulation of endothelial siveness to adenosine-induced contraction which corre- cells. ATP and Ap3A are potent dilator compounds of lates with a decreased expression of the A1 receptors. glomerular capillaries and their activity is mediated via These findings suggest that a purinergic mechanism signaling pathway P2Y receptor — NOS activity — cyclic may be involved in the regulation of glomerular GMP accumulation. Moreover, ATP and Ap4A and hemodynamics and subsequently the glomerular filtra- Ap5A induce contraction of intact glomeruli via activation rate. 186 Session 6. Metabolism of purines and pyrimidines in health and disease 2003

330 Oral Presentation Search for adenosine releasing agents for therapeutic applications Jerzy Barankiewicz Molecular Research Center, Inc., 5645 Montgomery Rd., Cincinnati, OH, USA

Adenosine (Ado) is a well recognized nucleoside that adenosine release and did not exhibit cardioprotective possesses a number of beneficial properties including, effects in clinical trialas. In in vitro studies cerebroprotective, anti-inflammatory and analgesic ac- AICA-riboside inhibited Ado uptake. tivities. Since elevation of Ado concentration can affect Another potential approach based on inhibition of many important physiological parameters, challenge to adenosine kinase (AK) has been also developed in pharmacologically manipulate Ado concentrations has Gensia Pharmaceuticals. Inhibition of AK could regu- attracted many laboratories, both pharmaceutical and late cellular Ado levels because AK actively phos- academic. phorylate Ado to AMP. Although it was reported that Since direct therapeutic uses of Ado are limited by se- AK inhibitors exhibit anti-inflammatory properties, no vere side effects such as hypotension and bradycardia, elevation of Ado in vitro or in vivo conditions has been as well as a short t 1/2, several concepts to regulate Ado detected. release were created and Ado regulating agents were More recently concept of “soft” adenosine deaminase studied. (ADA) inhibitors has been developed in Cypros Phar- First concept to regulate pharmacologically Ado con- maceuticals. It has been well documented that inhibi- centrations in heart and brain was developed in Gensia tion of ADA results in high elevation of Ado. In contrast Pharmaceuticals. The concept was based on the reports to highly toxic irreversible ADA inhibitors, “soft” re- that AICA-riboside (Acadesine) introduced to a dog be- versible ADA inhibitors potently elevate Ado and exert fore and during myocardial ischemia increased coro- cardio- and cerebroprotective effects. Use of EHNA and nary venous Ado concentrations, enhanced collateral its analogs may be a promising approach to cyto- blood flow, decreased arythmias, and inhibited protection of ischemic tissues, but more animal and granulocyte trapping. However after years of studies, it clinical studies is required. has been found that AICA-riboside did not stimulate

331 Oral Presentation Tissue specific alteration in expression level of adenosine receptors in streptozotozin-induced diabetic rats Marzena Grdeñ, Tadeusz Pawe³czyk Zak³ad Medycyny Molekularnej, Akademia Medyczna w Gdañsku, ul. Dêbinki 7, 80-211 Gdañsk

Adenosine is a nucleoside with great biological activ- a reference template. Obtained data demonstrated that ity able to affect cardiovascular, respiratory, nervous, mRNA level of A1 receptor with respect to values ob- excretory or immune systems. The action of adenosine tained in age- and sex-matched nondiabetic rats was is mediated by specific receptors (A1, A2a, A2b, A3) lo- 2-fold increased in diabetic kidney and was unchanged calized on plasma membranes. Change of tissue sensi- in diabetic heart and liver. The expression of A2b recep- tivity to adenosine is a common feature of diabetes and tor was decreased in diabetic kidney by 21% and was these alterations are cell and tissue specific. It may be unchanged in diabetic heart and liver. The level of A2a assumed that changes in tissue sensitivity to adenosine mRNA was decreased by 43% in diabetic liver, whereas may result from alterations in adenosine receptors. in diabetic heart and kidney expression of this receptor Therefore, the aim of our study was to investigate the was unchanged. The mRNA level for A3 receptor was expression level of adenosine receptors in tissues of di- not altered in diabetic heart, liver and kidney. abetic rats. In conclusion, our data indicate that the expression of The mRNA level of adenosine A1, A2a, A2b and A3 re- A1, A2a and A2b receptor is altered in diabetic rats in a ceptors in rat kidney, liver and heart was based on re- tissue specific manner, and that may lead to changes in sults from multiplex PCR performed with beta-actin as tissue sensitivity to adenosine. 2003 39th Meeting of the Polish Biochemical Society 187

332 Oral Presentation Effect of selected cardio-vascular drugs on serum adenosine deaminase activity Maria Kopff1, Edward Kowalczyk2, Anna Kopff3 1 — Zak³ad Chemii i Biochemii Klinicznej Wydzia³ Fizjoterapii, Uniwersytet Medyczny w £odzi, Pl. Hallera 1, 90-647 £ódŸ, 2 — Zak³ad Biofizyki i Fizjologii Cz³owieka Wydzia³ Wojskowo-Lekarski, Uniwersytet Medyczny w £odzi, Pl. Hallera 1, 90-647 £ódŸ, 3 — Klinika Kardiologii IMW, Uniwersytet Medyczny w £odzi, £ódŸ

In recent years potent cardioprotective properties of (at the dose of 6.42 mg/kg of body mass), acetylsalicylic several endogenous agents released from endothelial acid (4.29 mg/kg of body mass), simwastatin (3.43 cells and myocytes have been shown. Adenosine is one mg/kg of body mass) and molsidomine (0.69 mg/kg of of these substances. It is not only a potent coronary rabbits body mass). The control group of rabbits was vasodilator, but also possesses platelet inhibitory activ- given water by gastric tube at the same time. ity and anti-neutrophil properties, which include inhibi- Activity of ADA was assayed by colorimetric method tion of neutrophil activites, superoxide generation and according to Giusti and Galanti using adenosine and adhesion of these cells to the endothelium. The in- deoxyadenosine as substrates, and expressed in U/l. crease in adenosine production (and/or release of it to Using adenosine as a substrate, we have found that the extracellular space) would activate antioxidant en- acetylsalicylic acid, metoprolol and simvastatin de- zymes. Adenosine released to the blood stream is rap- creased ADA activity by about 44%, 29% and 50%, re- idly decomposed and has very short half-life time in hu- spectively. When deoxyadenosine was used as a sub- man blood. At the low concentrations adenosine is strate, the ADA activity decreased by 53% after phosphorylated to AMP (by adenosine kinase) and at acetysalicylic acid, by 51% after metoprolol, and by 63% high concentrations deaminated to inosine (by after simvastatin administration. Only molsidomine adenosine deaminase). administration did not change the activity of ADA. It The aim of this study was to determine the effect of seems that metoprolol, acetylsalicylic acid and drugs frequently applied in cardio-vascular diseases on simvastatin decreased activity of ADA and in conse- catabolism of adenosine, evaluated by adenosine quence prolonged the T 1/2 of adenosine what can ex- deaminase (ADA) activity measurement. We deter- plain in part the beneficial effect of these drugs. mined activity of ADA in serum of rabbits after three The study was financed by the Medical University in £ódŸ weeks daily intragastric administration of metoprolol through the intramural grant No.502-17-018.

333 Oral Presentation The activity of thymidine phosphorylase as a new ovarian cancer marker El¿bieta Miszczak-Zaborska1, Katarzyna Wójcik-Krowiranda2, Robert Kubiak3, Andrzej Bieñkiewicz2, Jacek Bartkowiak1 1 — Zak³ad Biochemii Lekarskiej, Instytut Fizjologii i Biochemii, Uniwersytet Medyczny, ul. Mazowiecka 6/8, 92-215 £ódŸ, 2 — I Klinika Ginekologii i Onkologii IGiP, Uniwersytet Medyczny, £ódŸ, 3 — Zak³ad Patologii Nowotworów, Katedra Onkologii, Uniwersytet Medyczny, £ódŸ

Thymidine phosphorylase (EC 2.4.2.4.) (TP) is in- A significantly higher TP activity was stated both in tu- volved in pyrimidine nucleoside metabolism, DNA syn- mor and in serum when compared to the control group. thesis and metabolism of pro-drugs of 5-fluorouracil and A high correlation was found between serum TP activity 5-fluorouracil. Its homology with the platelet-derived en- and tumor TP activity. Neoangiogenesis is higher in dothelial cell growth factor (PD-ECGF), a potent ovarian cancer, when compared to the group of border- angiogenic factor, has been shown. The activity of line malignancy tumors, but no correlation between thymidine phosphorylase was assessed by the spectro- IMD and TP activity in ovarian cancer was observed. TP photometric method in the cytosol of epithelial ovarian activity is significantly higher in more advanced neoplas- cancer tissues after surgery and in the pre-surgery se- tic lessions (FIGO III, FIGO IV) although no correlation rum from the same patients.The activity of TP in ovar- between TP activity and grading or histopatological type ian tumor and in serum was compared to the control of epithelial ovarian cancer was observed. group. Intratumoral microvessel density (IMD) was eval- Conclusion: Thymidine phosphorylase activity evalu- uated in tumor using immunohistochemical methods. ation might be useful in diagnostics of ovarian cancer. 188 Session 6. Metabolism of purines and pyrimidines in health and disease 2003

334 Oral Presentation Selective screening for genetic purine/pyrimidine disorders Ewa Pronicka, W. Gradowska, J. Sykut-Cegielska, M. Po³owska Department of Metabolic Diseases, Children’s Memorial Health Institute, Warsaw

Inherited disorders of purine and pyrimidyne (P/P) genase (DPYD) deficiency, dihydropyrimidinase metabolism have a wide variety of clinical symptoms, (DPYS) deficiency, ureidopropionase deficiency, often non-specific, among them: neurological (ataxia, thymidine phosphorylase deficiency (TP, MNGIE), dis- autism, epilepsy, deafness, dystonia, mental retarda- orders connected with mtDNA depletion. tion, muscle cramps and wasting, spasticity), small Screening methods set should allow to identify sev- stature, nephrolithiasis, recurrent infections, immuno- eral metabolites responsible for respective metabolic deficiency, macrocytic and hemolytic anaemia, mito- defects. Beside simple tests such as uric acid and chondrial dysfunction. The international proficiency orotate, the general screening system involves separa- programme ERNDIM recommends since last year to in- tion of the bases and nucleosides in urine (and clude P/P detection methods to screening procedures nocleotides in erythrocytes) by reversed-phase for inborn errors of metabolism. HPLC/UV with diode array detection. Several other To known diseases that require identification belong techniques are available (CE, LC-MS, GC-MS, NMR, over 25 various disorders connected with P/P metabo- tandem MS, TLC) allowing identification in urine, se- lism, such as: adenylosuccinate lyase (ADSL), rum or erythrocytes of P/P profile. Enzymatic and mo- phosphoribosyl pyrophosphate synthetase (PRPS) lecular methods verify positive screening results. superactivity, muscle adenosine monophosphate National selective screening programme performed deaminase (AMNPD1) deficiency, familial juvenile in Child Health Centre is currently implemented to P/P hyperuricaemic nephropathy (FJHN), xanthine diagnosing. Up to now number of such diagnoses is lim- dehydrogenase and sulfite oxidase (XDH, SO), heredi- ited, because of unsatisfactory knowledge among physi- tary orotic aciduria (UMPS), pyrimdine 5’-nucleotidase cians. deficiency (UMPH), dihydropyrimidinase dehydro-

335 Oral Presentation AMPD1 gene mutations — metabolic and clinical consequences of AMP deaminase deficiency Krzysztof Safranow Katedra i Zak³ad Biochemii i Chemii, Pomorska Akademia Medyczna, ul. Powstañców Wlkp. 72, 70-111 Szczecin

AMP deaminase (AMPD) converts AMP to IMP re- longer survival of patients with congestive heart failure leasing ammonia. AMPD activity is highest in skeletal (CHF) was observed in a retrospective study. Another muscles, where isoenzyme M encoded by AMPD1 gene prospective study showed significantly lower dominates. Myocardial AMPD activity is approxi- cardio-vascular mortality rate in C/T heterozygotes mately 100-fold lower. with ischemic heart disease (IHD). Muscle AMPD deficiency has been correlated with Proposed mechanisms of beneficial influence of features of myopathy since its first description in 1978. AMPD1 mutation usually take into account the role of AMPD1 mutation C34T in exon 2 responsible for most adenosine, which is produced by action of cases of deficiency was reported in 1992. Frequency of 5’-nucleotidases on AMP. AMPD deficiency associated mutant allele in Caucasian population is about 13%, re- with decreased AMP deamination might increase AMP sulting in 23% heterozygotes and 1.7% homozygotes. dephosphorylation rate, leading to synthesis of Other rare AMPD1 mutations were also found in exons adenosine. Its action through A1-A3 receptors in- 5 (G468T), 9 (C1162T) and 10 (G1274A). creases resistance of myocardium to ischemia (precon- Frequency of homozygotic C34T mutation in patients ditioning), protects against arrhythmias and inhibits with myopathy is not significantly higher than in production of TNF-a which plays major role in CHF healthy population. Therefore AMPD1 deficiency is of- decompensation. ten regarded now as a harmless metabolic variant. Post-exercise rise in skeletal muscle adenosine concen- The interest in AMPD1 was raised again in 1999 tration was observed in mutant homozygotes. when association of heterozygotic C34T mutation with Adenosine could be released into blood and reach 2003 39th Meeting of the Polish Biochemical Society 189 myocardium, but short half-life time in plasma (1–2 s) Due to high frequency of heterozygotes in Caucasian calls in question the possibility of direct influence of population, analysis of AMPD1 genotype may play im- adenosine produced in skeletal muscle on heart muscle. portant role in prognosis and treatment of patients. Ex- Increased adenosine production in heart of C/T patients planation of relationship between mutation and clinical was also suggested, but the expression of AMPD1 gene course of CHF and IHD should contribute to better un- in human myocardium is not well documented. derstanding of AMPD function.

336 Oral Presentation Decrease in daily urine excretion nad metabolites in patients with chronic renal failure Ewa S³omiñska1, Kamil Bury1, Przemys³aw Adamski1, Ryszard Smoleñski1, Przemys³aw Rutkowski2, Julian Œwierczyñski1 1 — Katedra i Zak³ad Biochemii, Akademia Medyczna w Gdañsku, ul. Dêbinki 1, 80-211 Gdañsk, 2 — Katedra i Klinika Nefrologii, Transplantologii i Chorób Wewnêtrznych, Akademia Medyczna w Gdañsku, ul. Dêbinki 7, 80-211 Gdañsk

NAD is an essential coenzyme for cellular red-ox reac- metabolism. The eluant from each fraction was injected tions and also a substrate for poly(ADP-rybosyl)ation. onto the liquid chromatography/mass spectrometry N-Methyl-2-pyridone-5-carboxamide (2PY) and system. Majority of 2PY and 4PY eluted in the first N-methyl-4-pyridone-5-carboxamide (4PY) are major fraction of this procedure. catabolites of nicotinamide which is released from Concentrations of 2PY in plasma of healthy subjects NAD in the process of poly(ADP-rybosyl)ation. and patients with chronic renal failure were 0.89 ± 0.18 Poly(ADP-rybosyl)ation plays a major role in cellular and 18.9 ± 3.6 mM, respectively while 4PY concentra- responce to DNA damage, increasing the plasma level tions were 0.35 ± 0.19 and 3.8 ± 1.2 mM in healthy sub- of NA (nicotinamide) and both 2PY and 4PY. Increase jects and patients with renal failure, respectively. An in plasma NAD metabolites is particularly important in average 24 h excretion of 2PY was 10.9 ± 2.9 and 5.2 ± patients with chronic renal failure since as much as 20 1.1 mmol/24 h in healthy subjects and patients with fold increase has been observed. It is uncertain, how- chronic renal failure, respectively. Similar decrease in ever, whether this increase is caused by DNA damage daily urine excretion was observed with 4PY: 2.7 ± 1.0 or by decrease of excretion with urine. The aim of this mmol/24 h and 1.4 ± 0.3 in healthy subjects and pa- study was therefore to estimate 24 h urine excretion of tients with renal failure, respectively. 2PY and 4PY in normal subjects and patients with re- We propose that decrease in daily urine excretion of nal failure. 2PY and 4PY is important factor responsible for in- 2PY and 4PY concentrations were measured using crease of plasma 2PY and 4PY concentrations in HPLC in urine from 24h collection, fractionated using uremic patients. procedure for evaluation of total human body purine

337 Poster Ectopurine metabolism and transport in glioma C6 cells Micha³ Komoszyñski, Ma³gorzata Romanowska Zak³ad Biochemii, Uniwersytet Miko³aja Kopernika, ul. Gagarina 9, 87-100 Toruñ

Astorcytes, besides neurones, blood platelets and en- nine in GC6 membranes. Contrary to synaptosomes, dothelial cells, are the important source of ecto-purines the glioma cells, under conditions inhibiting the Ado in brain. However, in astrocytes the metabolism and transport, degraded ecto-ATP with the final product be- transport of ecto-purines is much less known than in ing adenine and not inosine. We found neither neurons. The reported research is focused on metabo- deaminase nor ecto-Ado kinase on the glioma cell mem- lism of ecto-ATP and ecto-adenosine in the glioma C6 branes. Ecto-Ado was metabolized to adenine by the hy- cells (GC6). We found the complete set of enzymes de- drolysis of N-glycoside bond and not phosphorolysis. grading ecto-ATP into adenine on the plasma mem- Therefore that process was catalyzed by the ecto-purine brane surface. The research also indicated the presence N-glycosidase. However, re-synthesis of nucleotides of protein transporters different for adenosine and ade- from adenine and Ado, transported into the cell, was 190 Session 6. Metabolism of purines and pyrimidines in health and disease 2003 catalyzed by different enzymes adenine ribosyltrans- inosine and 2’-deoxyadenosine, whereas cytidine, ferase and adenosine kinase, respectively. Ecto-purine uridine and 2’-deoxyinosine did not affect the enzyme N-glycosidase effectively metabolized Ado at nano- activity. The strongest inhibitors of Ado and adenine molar and low micromolar concentrations (Km [Ado]= transport were dipyridamole and papaverine, respec- 1 mM; Vmax= 22.75 pmole/min/million cells for ade- tively. Km for Ado transporters was 9.8 mM and nine). The optimal pH for that enzyme is 7.0. The en- Vmax=3.62 pmole Ado/s/million cells. The above re- zyme was not activated by the divalent cations or phos- search indicates that the metabolism of ecto-ATP and phate. The Ado hydrolysis was inhibited by guanosine, ecto-Ado in neurons and glioma cells differs.

338 Poster Comparative research concerning behavior of GDP and AlF3 in vacuum and in water environments using molecular modeling Zygmunt Machoy1, Izabela Gutowska2, Jerzy Straszko3, Bogus³aw Machaliñski2 1 — Zak³ad Biochemii i Chemii, Pomorska Akademia Medyczna, ul. Powstañców Wlkp., Szczecin, 2 — Zak³ad Patologii Ogólnej, Pomorska Akademia Medyczna, ul. Powstañców Wlkp.72, Szczecin, 3 — Zak³ad Chemii Fizycznej, Politechnika Szczeciñska, Szczecin

The fluorine-aluminum compounds have been investi- groups and disrupts the P–O bond. Such a two-stage gated with increasing interest nowadays. From medical mode of reaction is confirmed by the calculations con- point of view, the neurotoxic properties of AlF3 are of cerning the length of the bounds, total energy E and the the most important, because of the recent reports con- molecule heat of formation. cerning its connection with pathogenesis of Alzheimer Our present research evaluated behavior of GDP and disease. AlF3 in water. The actual results are different from The aim of this study was to investigate the interac- those of previous studies performed in vacuum. AlF3 tion between aluminum fluoride (AlF3) and GDP. We does not disrupt the O–P bond in the phosphate group, used PM3 method with Polak-Ribier optimalization. but it binds to GDP forming GDP-AlF3 complex. AlF3 According to our previous results obtained in vac- binds with GDP interacting with a and b phosphates, uum, the main sites of the reaction in GDP are phos- although according to our calculation, binding with the phate groups. AlF3 can attack GDP between a and b b-phosphate residue leads to a more stable complex. Re- phosphate residues, although according to our calcula- sults obtained in water confirmed the calculations con- tions, the priority has the outer one. Computer simula- cerning the length of the bonds, total energy E and the tions with PM3 molecular modeling confirmed that the molecule heat of formation and they are consistent GDP molecule is firstly attacked by a single F– ion from with suggestions on the formation of GDP-AlF3 com- the AlF3 molecule. This ion reaches the phosphorus in plex in vivo, proposed by others. These results point the the phosphate group and pulls it away from the GDP importance of the conditions at which GDP interacts 2+ molecule (the O–P bond is stretched). Next, the AlF with AlF3. ion attacks the oxygen atom linking two phosphate

339 Poster AMP-deaminase from human placenta — effect of pH changes on kinetic and regulatory properties of the enzyme Gabriela Nagel-Starczynowska1, Anna Œwieca2, Anna Koryziak1, Ma³gorzata Œwi¹tkowska-Freund3, Jerzy Klimek1, Krystian Kaletha2 1 — Katedra i Zak³ad Biochemii Farmaceutycznej, Akademia Medyczna w Gdañsku, ul. Dêbinki 1, 80-211 Gdañsk, 2 — Katedra i Zak³ad Biochemii, Akademia Medyczna w Gdañsku, ul. Dêbinki 1, 80-211 Gdañsk, 3 — Instytut Po³o¿nictwa i Chorób Kobiecych, Klinika Po³o¿nictwa, Akademia Medyczna w Gdañsku, ul. Kliniczna 1a, 80-402 Gdañsk

AMP-deaminase (EC 3.5.4.6.; AMP-aminohydrolase, AMPD) tion of the purine nucleotides pool and stabilization of is a cytoplasmic enzyme widely found in animal tissues. The en- the adenylate energy charge (AEC). zyme catalyzes hydrolytic deamination of adenylic acid (AMP). Various isoforms of AMPD have been found in human The phisiological role of the enzyme is connected with regula- tissues. In skeletal muscle, activity of the enzyme is 2003 39th Meeting of the Polish Biochemical Society 191 much higher than that found in other non-(skeletal) and Pi), in the presence and in the absence of 100 mM muscle tissues. Recent inmmunological experiments potassium chloride. ATP activated the enzyme at each showed that the AMP-deamimase undergoes expres- pH; most significantly at pH 7.0. Similar profiles of reg- sion also in human term placenta. Physiological func- ulatory effect were observed in the presence of ADP tion of the enzyme in this tissue is not known. In this and GTP. In contrast, orthophosphate inhibited activ- work the influence of pH (6.0, 6.4 and 7.0) on activity ity of the enzyme at all pH. and regulatory properties of AMP-deaminase isolated The results presented here indicate that activity of from human placenta was investigated. Experiments AMP-deaminase is highly regulated and depends were performed both in the presence and in the ab- strongly on tested pH values. sence of important allosteric effectors (ATP, ADP, GTP

340 Poster Homocysteine-thiolactone hydrolase from yellow lupin seeds; purification to homogene- ity Ma³gorzata Pietrowska-Borek1, El¿bieta Starzyñska2, Andrzej Guranowski2 1 — Department of Plant Physiology, University of Agriculture, ul. Wo³yñska 35, 60-637 Poznañ, 2 — Department of Biochemistry and Biotechnology, University of Agriculture, ul. Wo³yñska 35, 60-637 Poznañ

Homocysteine-thiolactone (HcyTL), a cyclic thioester Continuing studies on the recently described of homocysteine, occurs in all organisms investigated. HcyTL-ase, partially purified from yellow lupin seeds It is synthesized by methionyl-, leucyl- and iso- [2], we have developed procedure that allows one to ob- leucyl-tRNA synthetases that misactivate Hcy forming tain this enzyme in electrophoretically homogeneous Hcy~AMP, from which Hcy is never transferred to state. This has been achieved by electrophoresis in t-RNA but edited as HcyTL. Due to energy stored in the polyacrylamide gel conducted under nondenaturating thioester bond, HcyTL modifies proteins post-trans- conditions. The HcyTL-ase eluted from the gels and lationally either by forming adducts in which the car- then subjected to SDS/PAGE showed a single band of boxyl-group of Hcy is linked by amide bonds to epsi- 32 kDa. Since the molecular mass of the native lon-amino group of protein lysyl-residues or by the HcyTL-ase, estimated by gel filtration, was approxi- Hcy-S-S- linkage to protein cysteinyl-residues. mately 60-kDa, we conclude that the lupin enzyme func- Homocysteinylation impairs protein structure and tions as a dimer. function. However, mammals [1] and plants [2] have References: enzyme activity that detoxifies HcyTL. 1. Jakubowski H (2000) J Biol Chem, 275: 3957–3962. 2. Jakubowski H, Guranowski A (2003) J Biol Chem, 278: 6765–6770.

341 Poster Effect of insulin on the expression level of nucleoside transporters in cultured rat cardiac fibroblasts Marzena Podgórska, Tadeusz Pawe³czyk Zak³ad Medycyny Molekularnej, Akademia Medyczna w Gdañsku, ul. Dêbinki 7, 80-211 Gdañsk

Cardiovascular disease is one of the main complica- the heart, adenosine level depends on the metabolism tions in diabetes. Adenine nucleotides and adenosine of that nucleoside and its transport across the plasma are important for the myocardial function. Adenosine membranes. has been proposed as a regulator of coronary blood The aim of this study was to investigate the effect of flow. Moreover, adenosine modulates the effects of insulin on expression level of the nucleoside transport- catechlamines on the heart and blocks myocardial slow ers in cultured rat cardiac fibroblasts. action potentials. Accordingly, processes that modify Rat cardiac fibroblasts were isolated using the colla- adenine nucleotide and adenosine metabolism result in genase perfusion technique. Cells were cultured for altered heart function. In particular compartment of 5 days in Dulbecco’s modified Eagle’s medium with insu- 192 Session 6. Metabolism of purines and pyrimidines in health and disease 2003 lin and in the presence of 25 mM glucose. The expres- tion of the cells to the insulin resulted in 60% decrease of sion levels of CNT1, CNT2, ENT1 and ENT2 in fibro- the mRNA level for CNT2. Insulin treatment had no ef- blasts of normal rats were examined by multiplex PCR fect on the expression level of nitrobenzylothioinosine with beta-actin as a reference template. Our studies sensitive transporter (ENT1) in cardiac fibroblasts. The showed the presence of mRNA for concentrative so- maximal change in the CNT2 mRNA level was observed dium-dependent nucleoside transporter (CNT2) and at8hofincubation with 10 nM insulin. equilibrative nucleoside transporters (ENT1, ENT2) in In conclusion, our data suggest that in diabetic car- the cultured fibroblasts. There was the same nucleoside diac fibroblasts there is the change in expression of transporters’ expression profile in isolated cardiac nucleoside transporter (CNT2). Such a change in CNT2 fibroblasts thus indicating that the culture conditions expression might contribute to increased concentra- did not cause the loss of nucleoside transporters. Exposi- tion of adenosine observed in the diabetic heart.

342 Poster Adenosine deaminase of pig brain synaptosomes Ma³gorzata Romanowska, Micha³ Komoszyñski Zak³ad Biochemii, Uniwersytet Miko³aja Kopernika, ul. Gagarina 9, 87-100 Toruñ

One of the key enzymes metabolizing adenosine (Ado) and 2 and 5’-nucleotidase). The synaptosomal ADA of is adenosine deaminase (ADA). In brain ADA occurs molecular weight 42.4 ± 1.1 kDa had no sugar groups. It mainly in cytosol, although its presence was detected deaminated adenosine and 2’-deoxyadenosine with sim- also on the surface of synaptosomes and neurons. Due ilar rate, revealed a broad pH optimum and was not ac- to its very low activity, function of ADA on the surfaces tivated by the metal ions. Its activity was inhibited by of the nervous system cells is unclear. In addition to EHNA (erytro-9-(2-hydroxy-3-nonylo)adenine). Dipyri- degradation of Ado to inosine and ammonium ions, damole, NBTI, inosine, 2’-deoxyinosine, cytidine, ecto-ADA seems to play another role, participating in guanosine, ATP, ADP and AMP had not affected the ac- adhesion and regulation of the A1 receptor activity. tivity. The enzyme revealed low affinity to substrates Adenosine is an important neuromodulator in the (Km=255 mM for adenosine and 290 mM for mammalian brain since it regulates the secretion of 2’-deoxyadenosine). The physiological concentration of neurotransmitters affecting P1 receptors, participates ecto-Ado in brain is 0.3–1000nM and may be even in apoptosis, cell proliferation and exocytosis of trophic 100-fold higher due to hypoxia. Therefore, ADA seems factors. We have detected the presence of ADA in not to play any significant role in the degradation of synaptosomes of pig hippocampus, medulla, cerebel- physiological concentrations of Ado and at higher con- lum and brain cortex. The synaptosomal activity of centrations, Ado is degraded with low rates. These re- ADA was significantly lower than that of other en- sults suggest that the basic mechanism of decreasing zymes of ecto-ATP degradation cascade (NTPDases 1 the Ado concentration in synapses might be a re-uptake mediated by specific protein transporters.

343 Poster Optimization of cellular and molecular assay with AMP deaminase for toxic and eco-toxic properties of various chemicals Izabela Rusiecka, Anna Ludwiczak, Konrad Kleszczyñski, Andrzej Sk³adanowski Zak³ad Enzymologii Molekularnej, Miêdzyuczelniany Wydzia³ Biotechnologii UG i AMG, ul. Dêbinki 1, 80-211 Gdañsk

The experiments were performed within the network systems. Biological systems, adapted for that purpose, of environmental laboratories within Europe under the involve: well characterised isolated proteins with spe- common project: “Designing molecular tests for toxico- cific functions, living cells, whole organisms including logical and ecotoxicologial risk assessment”. animals, multi-species tests, micro- and meso-cosms. Newly synthesized compounds due to legislation have The aim of this study was to elaborate the back- to undergo verification of their toxic and eco- toxic ground for cost-effective and efficient screening pro- properties using variety of standardized biological test cedure for existing and new compounds using both cel- 2003 39th Meeting of the Polish Biochemical Society 193 lular (HeLa cells) and molecular assay (AMP-deami- Having employed the WST-1 test for cytotoxicity in nase activity). HeLa cell culture, a variety of ionic solvents was checked The tested compounds were ionic amphiphillic solvents and structure-activity relationship for various lengths of of extremely low vapour pressure used in organic synthe- hydrocarbon chain in ionic solvents estimated. sis and synthetic musks added to household products. [DMIM][BF4] with 10 carbons (3-butyl-1- 3-methyloimi- The enzyme purified from skeletal muscle appeared to dazolium tetrafluoroborate) appeared to be the stron- be a very sensitive indicator of inhibitory properties of gest cell death initiator. AMP deaminase isolated from both groups of substances. The strongest effect was ex- the HeLa cell culture was inhibited by analogous com- erted by musk ketone; with Ki=0.15 mM. Almost all ionic pounds but the effect was less pronounced than in the solvents inhibited AMP deaminase. The survey has been case of the skeletal muscle enzyme. made using 96-well plates with immobilized enzyme to In summary, we propose the AMP deaminase inhibi- screen ionic solvents in a multi-sample manner. This mod- tion assay as a useful element of the testing battery for ification was proved compatible with the reaction in liquid chemical compounds that can potentially accumulate phase. and interfere with environment.

344 Poster Properties of AMP-deaminase in ontogenesis of human hearts Iwona Rybakowska1, Ma³gorzata Œwi¹tkowska-Freund2, Janusz Emerich3, Ma³gorzata Kortecka4, Maciej Krzy¿anowski5, Krystian Kaletha1 1 — Katedra i Zak³ad Biochemii, Akademia Medyczna w Gdañsku, ul. Dêbinki 1, 80-211 Gdañsk, 2 — Instytut Po³o¿nictwa i Chorób Kobiecych, Klinika Po³o¿nictwa, Akademia Medyczna w Gdañsku, ul. Kliniczna 1a, 80-402 Gdañsk, 3 — Instytut Po³o¿nictwa i Chorób Kobiecych, Klinika Ginekologii, Akademia Medyczna w Gdañsku, ul. Kliniczna 1a, Gdañsk, 4 — Instytut Po³o¿nictwa i Chorób Kobiecych, Klinika Neonatologii, Akademia Medyczna w Gdañsku, ul. Kliniczna 1a, Gdañsk, 5 — Katedra i Zak³ad Medycyny S¹dowej, Akademia Medyczna w Gdañsku, ul. Dêbinki 7, Gdañsk

AMP-deaminase is a highly regulated enzyme, cata- pose that similar pattern of differentiation of lyzing irreversible deamination of adenylic acid AMP-deaminase may also take place during human (5’-AMP), located at branchpoint of adenylate nucleo- ontogenesis. In the presented work we examined and tide catabolism. It plays an important role in stabilisa- compared some physiochemical properties of the en- tion of adenylate energy charge (AEC) and regulation zyme isolated from 6 months-old fetus, one-month-old of purine nucleotide pool in the cell. infant and adult human hearts. Chromatography on Investigations of Holme’s group showed the presence phosphocellulose revealed existence of two molecular of three developmental forms of AMP-deaminase (em- forms of the enzyme at all stages of human heart devel- bryonic, perinatal and adult) in growing rat skeletal opment. The enzyme form that was eluted by 0.75 M muscle. Research on human skeletal muscle showed KCl constituted about 10% of the total activity. also differences in kinetic and chromatographic proper- AMP-deaminase isolated from heart of one-month-old ties of developmental forms of AMP-deaminase. Re- infant showed higher activity than that observed for semblance of structural and immunological features be- the counterparts isolated from hearts at two other de- tween human and rat muscle isoenzyme permits to sup- velopmental stages.

345 Poster Purine nucleosides and oxypurines in porcine myocardium and coronary sinus during reperfusion Krzysztof Safranow1, Monika Raæ1, Katarzyna Jakubowska1, Ryszard Rzeuski2, Mariusz Listewnik2, Maria Olszewska1, Dariusz Chlubek1 1 — Katedra i Zak³ad Biochemii i Chemii, Pomorska Akademia Medyczna, ul. Powstañców Wlkp. 72, 70-111 Szczecin, 2 — Klinika Kardiologii, Pomorska Akademia Medyczna, ul. Powstañców Wielkopolskich 72, 70-111 Szczecin

Myocardial levels of ATP, adenylate energy charge quence leads to dephosphorylation of nucleotides and (AEC), nucleosides and oxypurines are commonly used increase in tissue concentrations of nucleosides and markers of energy state. Ischemia-reperfusion se- oxypurines. Measurement of their concentrations in 194 Session 6. Metabolism of purines and pyrimidines in health and disease 2003 coronary sinus blood is much easier than in human reaching the basal values at 120’. Changes of mHyp myocardium. concentrations (0.41, 1.09, 0.97, 0.68, 0.60) were rela- The aim of the study was to assess how changes in cor- tively smaller. onary sinus plasma concentrations of hypoxanthine CsIno (9.65, 21.85, 14.65, 11.65, 11.99) correlated and inosine reflect changes in myocardial purines dur- positively with mIno (+0.56), mAdo (+0.56) and mHyp ing reperfusion following cardioplegic ischemia. (+0.52). CsHyp (36.7, 51.0, 46.6, 46.5, 48.3) correla- 24 pigs weighing 48–66 kg were subjected to open tions with mHyp (+0.47), mIno (+0.40), mAdo (+0.35) heart surgery with 1 hour of cold cardioplegic arrest and were weaker. Median (cs-a)Ino profile (–1.12, 1.13, additional dose of warm cardioplegia. The samples of –0.42, –1.69, –1.21) was parallel to mIno, but individ- myocardium (m) from left ventricular apex and blood ual values showed greater variance and correlations from coronary sinus (cs) and aorta (a) were taken in the with mAdo (+0.19) and mIno (+0.14) were weaker than pre-ischemic period, prior to aortic cross-clamping (t0), for csIno. Changes in (cs-a)Hyp concentrations (4.39, than at 5’, 20’, 60’ and 120’ of reperfusion. Purine levels 4.79, 4.36, 4.08, 4.09) were not significant. Negative in myocardium specimens (nmol/mg protein) and correlations between mAEC and csHyp (–0.28) or plasma (micromol/L) were measured using HPLC. The csIno (–0.25) were significant but weak. difference between concentrations in coronary sinus Conclusions: Inosine concentration in coronary sinus and aorta (cs-a) was calculated for each sample. plasma seems to be the optimal marker of purine Median mIno (0.36, 5.02, 2.75, 0.77, 0.42) and mAdo nucleoside accumulation in myocardium during (0.034, 1.21, 0.24, 0.057, 0.028) showed similar ischemia. None of the investigated sinus plasma param- changes in subsequent biopsies (t0, 5’, 20’, 60’, 120’). eters was a good predictor of myocardial adenylate en- They were maximal at 5’ and subsequently decreased, ergy charge.

346 Poster Relationship between ATP release and its extracellular metabolism in isolated rat glomeruli

Joanna Soboñska1, Gabriela Dzier¿ko2, Ludmi³a Martyniec1, Stefan Angielski1 1 — Laboratorium Nefrologii Komórkowej i Molekularnej PAN, Centrum Badañ Medycznych, Polska Akademia Nauk, Gdañsk/Warszawa, 2 — Katedra i Zak³ad Analityki Klinicznej, Akademia Medyczna w Gdañsku, ul. Dêbinki 7, 80-041 Gdañsk

Recently we have shown that extracellular ATP in- pected inhibitory effect of AOPCP on ATP release duces relaxation of glomerular microvasculature by ac- could be due to the direct effect of AMP on the process tivating the P2Y purinoreceptors. In the present study of ATP release from glomeruli. It has been shown that we have investigated release of ATP from isolated AMP also strongly inhibits ATP release (about 70%). glomeruli and its extracellular metabolism. IMP (1mM) had no effect on ATP release. The inhibi- Glomeruli were isolated from the renal cortex of adult tory effect of AMP on ATP release was significantly at- male Wistar rats.The minced cortex was mashed tenuated in the presence of Ap5A (inhibitor of through a gradual sieve. The extracellular ATP concen- ecto-adenylate kinase). The addition of ADP (10 mM) tration was quantitated by luminometric luciferin-lu- to isolated glomeruli caused an accumulation of ATP ciferase assay. Samples (50 ml) were boiled for 2 min. that was also blocked by Ap5A (5 mM). The present and centrifuged to eliminate possible cell contaminants. study provides evidence for ATP release from ATP release from isolated glomeruli was 0.9 glomeruli and demonstrates that the decrease in pmol/h/thousand glomeruli. ARL (50 mM) inhibitor of extracellular ATP observed after addition of either ecto-ATPase increased extracellular concentration of AOPCP or AMP is caused by ecto-adenylate kinase ac- ATP from 0.9 to 1.6 pmol/h/thousand glomeruli. In tivity in rat glomeruli. It is known that cytosolic the presence of 50 mM AOPCP (inhibitor of ecto-5’-nu- adenylate kinase catalyses the near-equili- cleotidase), ATP release was decreased by 57%. The brium-reactions and even small rise of AMP concen- level of ATP in tissue remained unchanged. The unex- tration leads to a large fall in concentration of ATP. 2003 39th Meeting of the Polish Biochemical Society 195

347 Poster Ap4A modifies renal function by P2 purinoreceptors Anna Stiepanow-Trzeciak, Aleksandra Rudzik, Miros³awa Szczepañska-Konkel Katedra i Zak³ad Analityki Klinicznej, Akademia Medyczna w Gdañsku, ul. Dêbinki 7, 80-041 Gdañsk

Extracellular diadenosine 5’5’’’-P1,P4-tetraphosphate fusion of b,g-Me-ATP (2 mmol/kg + 20 nmol/kg/min) (Ap4A) can be involved in the depression of renal increased: urine excreation from 10.2 ± 2.2 to 40.2 ± 2.5 hemodynamics. Vascular effects induced by Ap4A have ml/min and sodium excreation from 0.6 ± 0.22 to 2.47 ± been reported to be mediated by P2 purinoreceptors 0.46 mmol/min, whereas RPF and GFR were (P2X and P2Y-subtypes). The effects of intravenous in- unaffacted. In contrast to b,g-Me-ATP, 2-MeS-ATP (2 fusion of Ap4A and ATP analogues b,g methylene-ATP mmol/kg + 20 nmol/kg/min) did not significantly mod- (b,g-Me-ATP) and 2-methylthio-ATP (2-MeS-ATP), as ulate urine excretion, but reduced GFR from 2.15 ± specific agonists of P2X and P2Y receptors, respec- 0.36 to 1.0 ± 0.21 ml/min and RPF from 9.8 ± 1.9 to 5.2 tively, on renal plasma flow (RPF), glomerular filtra- ± 0.93 ml/min. 3. Pretreatment with nonselective tion rate (GFR) and urinary sodium excretion were de- P2-purinoreceptor antagonist, suramin (12 mg/kg + termined in the anesthetized Wistar rats. RPF and 0.18 mg/kg/min) completely abolished the effects of GFR were measured as a clearance of p-amino- Ap4A on GFR, diuresis and natiuresis, whereas pre- hippurate and radioactive inulin, respectively. treatment with selective P2Y-purinoreceptor antago- Results: 1. Infusion of Ap4A (2 mmol/kg + 20 nists, RB-2 (10 mg/kg +0.1 mg/kg/min) abolished in- nmol/kg/min) decreased RPF from 10.4 ± 1.9 to 5.3 ± hibitory effect of Ap4A on GFR. 0.95 ml/min and progressively GFR from 2.19 ± 0.3 to Conclusion: Diuretic and natiuretic effects of Ap4A 1.0 ± 0.09 ml/min. In spite of reduction of GFR the sig- are probably mediated by tubular P2X-purinoreceptors, nificant increased in sodium and urine execreation whereas renal hemodynamics by vascular P2Y-subtype were observed: 0.5 ± 0.09 vs. 2.0 ± 0.09 mmol/min and purinoreceptors. 10.2 ± 2.1 vs. 27.5 ± 3.2 mmol/min, respectively. 2. In-

348 Poster Calcium strongly activates guanosine nucleosidase from yellow lupin (Lupinus luteus) seeds Maciej Szuwart, A. Guranowski Department of Biochemistry and Biotechnology, University of Agriculture, ul. Wo³yñska 35, 60-637 Poznañ Guanosine nucleosidase discovered by us in the ex- sis measured in the absence of that salt. Half stimula- tracts of yellow lupin seeds has been purified to homo- tion was observed at 50 mM calcium chloride. Hydroly- geneity. Its molecular mass amounts 80 kDa, optimum sis of inosine and adenosine, that also are substrates of pH 4-5, and Km for guanosine 4.5 mM. During enzyme the nucleosidase, is stimulated by calcium, too; al- characterization, it has been found that among various though to smaller extend. Many other ions did not af- anions and cations investigated calcium ion is the only fect the course of guanosine hydrolysis. However, am- one that strongly stimulates the hydrolysis of monium molybdate, copper sulfate, sodium sulfite, so- guanosine to guanine and ribose. dium periodate, and manganese, zinc and copper chlo- At 2.5 mM concentration, calcium chloride stimu- rides acted as inhibitors. lated the reaction six-fold, when compared to the cataly-

349 Poster Variable expression of cytosolic 5’-nucleotidase in human skeletal muscles Kinga Tkacz-Stachowska1, Katarzyna Lechward2, Andrzej Sk³adanowski1 1 — Zak³ad Enzymologii Molekularnej, Miêdzyuczelniany Wydzia³ Biotechnologii UG i AMG, ul. Dêbinki 1, 80-211 Gdañsk, 2 — Department of Functional Genomics, Tokyo Medical and Dental University, 1-5-45 Yushima Bunkyo-ku, 115-8510 Tokyo, Japan Cytosolic 5’-nucleotidase type I (cN-I) belongs to solu- kinetic parameters and localisation in the cell. cN-I is ble 5’-nucleotidases that differ in substrate preference, the major isoenzyme which prefers AMP as a substrate 196 Session 6. Metabolism of purines and pyrimidines in health and disease 2003 and is responsible for adenosine production during im- pressed in human skeletal muscles. The highest level of balance between oxygen supply and work demand; like cn1a was found in muscles classified mostly as during ischemia or prolonged intensive energy output. slow-twitch oxidative ones (m. quadriceps femoris and Two cn1 loci exist in the human genome, namely cn1a m. gluteus maximus). On the other side, expression was and cn1b located on chromosomes 1 and 2, respec- significantly lower in such muscle like m. tibialis ante- tively. cn1a transcripts were found in most of human rior, m. brachioradialis, m. deltoideus and m. biceps tissues, and the highest RNA levels were observed in brachii. The amount of 40 kDa protein recognized by the heart, skeletal muscle, brain and testes. anti cN-Ia antibodies usually correlated with the tran- The aim of this study was to measure expression of script level. cn1 genes in human skeletal muscles varying in the Specimens of m. quadriceps femoris showed also sig- level of oxygen metabolism and to correlate it with the nificant individual variability in myoglobin gene ex- expression of myoglobin gene as an oxidative metabo- pression indicating differences in the degree of oxygen lism marker. metabolism in particular samples. The latter has been Expression of cn1a was evaluated by Northern and weakly positively correlated (corr = 0.51) with cn1a ex- Western blotting in 38 samples of six different types of pression. skeletal muscles taken during surgery. In order to ver- We conclude that the more complexed type of depend- ify expression of cn1b we adapted RT-PCR technique ence exists between expression of cn1a and myoglobin and Western blotting. genes however the oxidative slow-twitch type of muscle The results showed substantial individual differences seems to be a primary place for active AMP-selective of cn1a gene expression whereas cn1b gene was not ex- 5’-nucleotidase.