- Email: [email protected]
Are succunylpurines neurotoxic?
POSTER ABSTRACTS
166 APRT DEFICIENT MICE A S AN I N VIVO MAMMALIAN MODEL FOR MUTATION Peter J. Stambrook, a n d J a y A. Tischfield, University of Cincinnati, College of Medicine a n d I n d i a n a University School of Medicine There are currently no satisfactory in vivo systems for monitoring the mutagenic capacity of drugs, chemicals or environments. We are proposing the use of APRT-deficient mice carrying m u t a n t but revertible A P R T alleles as a mutagenesis model. The basis for the model is the differential capacity o f A P R T + and A P R T cells to metabolize exogenous adenine. Cells t h a t are A P R T ÷ will convert labeled adenine to labeled AMP a n d ultimately incorporate the tagged adenine derivative into nucleic acids. Cells t h a t are A P R T - will not. Objectives To determine w h e t h e r or not rare A P R T + cells (analogous to APRT + revertants) can be detected in a n A P R T background in vivo following exposure to 14C-adenine. Methods Embryonic stem (ES) cells t h a t are tumorigenic were r e n d e r e d APRT by targeted homologous recombination and subsequent selection in 2,6 diaminopurine (DAP). These APRTES cells were mixed with APRT ~ wild type ES cells at ratios of 10:1 a n d 50:1 (APRT /APRW.) a n d 5 × 106 cells were injected into each flank of a n A P R T - syngeneic mouse. Tumors were allowed to grow to 0.5-1.0 cm, at which time mice were injected I.P. with 80 ~Ci ~4C-adenine (4 ~Ci/gm wt). After 48 hours, the tumors were surgically removed, and fixed and sectioned, a n d coated with emulsion for autoradiography. Results Autoradiograms show t h a t cells in the t u m o r become labeled in proportion to the fraction o f A P R T ÷ cells in the initial innoculum. Some cells h a d nuclear and cytoplasmic labeling, indicative of DNA replication during 14C-adenine exposure. Other cells exhibited cytoplasmic labeling only. Tissues from the A P R T - host mouse lacked radioactive cells. Conclusions I.P. injection of 14C-adenine in A P R T - mice with revertible A P R T alleles will be sufficiently sensitive to detect rare A P R T + cells.
167 MODULATION OF DEOXYCYTIDINE KINASE SUBSTRATE SPECIFICITY IN LYMPHOCYTE Staub M., Keszler G., Sasv~ri-Sz6kely M. and Spasokukotskaja T., Semmelweis University of Medicine, Dept. of Med. Chem, Mol. Biol. a n d Pathobioehem., Budapest, Hungary Deoxycytidine kinase (dCK) h a s a broad s u b s t r a t e specificity, responsible to phosphorylate dCyd, dAdo, rAdo and their analogues used in therapy. The potentiation o f d C K activity h a s been shown after short t e r m t r e a t m e n t with 2-Cl-dAdo (CdA) in h u m a n tonsillar lymphocytes a n d a post-translational modification of the enzyme was suggested (1995 ESPPM, Vasta, Italy). In addition to tonsils, the "self-potentiation" by CdA was also found in h u m a n peripheral blood mononuclear cells, HL-60 cells, mouse thymocytes a n d spleen cells. The degree of stimulation was higher in G-phase, as in S-phase enriched cells. Not only CdA, but a serie of other toxic analogues can induce the potenciation of the kinase, responsible for t h e i r phosphorylation, while thymidine kinase (TK) is not affected. Even the n a t u r a l nucleoside dAdo, h a s increased the activity of dCK, if its d e a m i n a t i o n was prevented by deoxycoformycin, The concentration dependence of dAdo on the activation of dCK is biphasic, similarly to receptor binding curve of Ado, suggesting the signal transduction in the process. Inhibitors of the signal t r a n s d u c t i o n p a t h w a y were tested, a n d NaF, the universal inhibitor of phosphatases, h a s also induced the increase of dCK activity. CLINICAL BIOCHEMISTRY, VOLUME 30, APRIL 1997
The s u b s t r a t e specificity of the "potentiated" dCK was changed, induced either by the toxic analogue (CdA), or by NaF. The affinity of the enzyme to dCyd h a s been increased, while to CdA it h a s been decreased, stimulated e t h e r by CdA or by NaF. The m e c h a n i s m producing the "potentiated" nucleoside kinase might be different, induced by the nucleoside or by NaF, b u t both processes probably involve signal transduction mechanisms.
168 THIOPURINE-METHYLTRANSFERASE ACTIVITY AND TOXICITY OF AZATHIOPR1NE IN RHEUMATOID ARTHRITIS J. Stolk, A. Boerbooms, R. De Abreu*, D. de Koning*, L. v a n de P u t t e Depts. of Rheumatology & Paediatrics*, University Hospital Nijmegen P.O. Box 9101, 6500 HB Nijmegen, The N e t h e r l a n d s Objective To investigate in rheumatoid arthritis (RA) w h e t h e r purine enzyme activities may be predictive for development of azathioprine (AZA) related severe toxicity. Design & Methods P a t i e n t s with longstanding RA (n= 33) entered a prospective study for t r e a t m e n t with AZA. Before s t a r t and at m o n t h 1 and 6 of t r e a t m e n t , we m e a s u r e d activities of thiopurinem e t h y l t r a n s f e r a s e (TPMT). Controls included patients with early RA before second line t r e a t m e n t and h e a l t h y volunteers. Results Of the 33 patients 14 developed toxicity, mainly gastrointestinal intolerance, in 11 necessitating to definite withdrawal of AZA within 5 weeks after start. P a t i e n t s with toxic adverse effects h a d significantly lower TPMT activities at s t a r t and at the time of withdrawal (P <- 0.004) a n d in 50% we found a baseline intermediate TPMT activity. Of the 8 patients with baseline intermediate TPMT levels, 7 developed toxicity. There was a significant relation between intermediate TPMT activity and side effects (P = 0.005). Compared to high activity, a baseline intermediate TPMT activity resulted in a Relative Risk of 3.1 (95% CI: 1.6-6.2) to develop severe toxicity on AZA. Conclusion In RA, a p r e - t r e a t m e n t intermediate TPMT activity is predictive for development of severe toxicity on AZA. As clinical implication one should consider TPMT m e a s u r e m e n t prior to treatment. Gastrointestinal side effects are not necessarily caused by 'hypersensitivity', but may be based on a (thio)purine metabolic disbalance.
169 ARE SUCCINYLPURINES NEUROTOXIC? Stone, T.W., Roberts, L.A., Morris, B.J., Jones, P.A., Duley, J.A. and Ogilvy, H.V., West Medical Building, University of Glasgow, Glasgow G12 8QQ, Scotland Objectives P a t i e n t s with a deficiency of adenylosuccinate lyase exhibit m e n t a l retardation, a u t i s m a n d epilepsy, with a n accumulation of two abnormal purines succinyladenosine (S-aden) and succinylaminoimidazole carboxamide ribotide (SAICAr) in the CSF. We have now tried to determine w h e t h e r these could produce neurotoxic effects in the brain. Design and Methods Each of the purines was applied initially by microinjection directly into the hippocampus of anaesthetised rats, and in later experiments by reverse microdialyses over a period of 10 hours. The solution injected or dialysed contained 2 mmolesfl of the abnormal purines. In b o t h cases, the animals were allowed to recover for 7 days, w h e n the b r a i n s were removed for histological analysis. Results The initial, single injections of each purine did not induce any signs of neuronal damage or degeneration, perhaps because the compounds were not present for a sufficiently long period. However, after reverse microdialysis, 5 of 6 animals receiving
283
9TH INTERNATIONAL/6TH EUROPEAN J O I N T SYMPOSIUM ON PURINE AND PYRIMIDINE METABOLISM IN MAN SAICAr showed significant degeneration of pyramidal neurones. S-aden h a d no effect. Conclusion SAICAr h a s a neurotoxic action on pyramidal neurones of the r a t hippocampus. If a similar action occurs elsewhere, and in h u m a n s , such neuronal damage could contribute to the m e n t a l retardation, a u t i s m a n d epilepsy found in some p a t i e n t s with adenylosuccinase deficiency.
170 SUCCINYLPURINES DO NOT MODIFY GLUTAMATE OR A D E N O S I N E EFFECTS IN THE CNS Stone, T.W., De Abreu, R.A., Duley, J.A., Gross, M., Salerno, C., v a n den Berghe, G.
Objectives To determine if the abnormal purines detected in patients suffering from deficiency of adenylosuccinate lyase, succinyladenosine (S-aden) a n d succinylaminoimidazole carboxamide ribotide (SAICAr) have any effect on synaptic transmission in the hippocampus. Design and Methods Slices 500 p,m thick were prepared from male rats a n d prepared for recordings of synaptic potentials evoked from the s t r a t u m r a d i a t u m a n d single cell firing rates. Results S-aden a n d SAICAr did not modify either the evoked population spike potential or the evoked population excitatory postsynaptic potential (glutamatergic) at concentrations up to 0.2 mM, the concentration found in the CSF of patients. These purines did not modify responses of single cells to g l u t a m a t e or several analogues. Adenosine itself depressed synaptic potentials, in the range of 5-50 ~LM. However S-aden or SAICAr did not modify, by blocking or enhancing, the effects of adenosine. Conclusion Neither of the abnormal purines modify synaptic transmission in the r a t hippocampus. It seems unlikely t h a t a disruption of synaptic transmission contributes substantially to the CNS symptoms of some p a t i e n t s with adenylosuccinase deficiency.
171 URINARY SCREENING FOR PYRIMIDINE METABOLISM DISORDERS: REFERENCE RANGES FOR DIHYDROURACIL, URACIL A N D DIHYDROURACI/URACIL RATIO Satoshi Sumi, Kiyoshi Kidouchi, Katsuo Hayashi, Masaynki Imaeda, Masami Asai, Yoshiro Wada
Objectives Pyrimidine chemotherapy agent such as 5-FU are used widely but can occasionally cause serious adverse reactions in patients with pyrimidine metabolism disorders. We have analyzed u r i n a r y pyrimidine levels in 167 h e a l t h y adults and 966 p a t i e n t s with malignancy, hypertension, cerebral infarction, etc. The reference range of dihydrouracil for h e a l t h y adults was 23.8 ± 35.5 (mean ± S.D.; ~mol/g creatinine) and t h a t of uracil was 63.3 -+ 66.0. T h a t ofdihydrouracil/uracil ratio was 0.31 ± 0.16. In addition, a n asymptomatic m a n with dihydropyrimidinuria was detected in this study. We performed a n oral uracil loading test on him. The uracil and the dihydrouracil level in his blood were extremely high after the test, suggesting t h a t the 5-FU level would be also high if it was treated.
enzymes of purine nucleotide biosynthesis have been determined or are currently being determined at high resolution by x-ray diffraction of crystals. The structures of a n u m b e r of enzymes of nucleobase salvage and catabolism have also been solved. These structures provide a wealth of information about mechanisms of catalysis, consequences of deleterious mutations in humans, and potential strategies for drug development. Valuable generalizations concerning enzymatic mechanisms of several reaction types t h a t are i m p o r t a n t in nucleotide metabolism, such as phosphoribosyltransfer, glutamine dependent amination, and a s p a r t a t e dependent amination, have emerged from the structural studies. These concepts will be illustrated with a n analysis of phosphoribosyltransferase enzymes based on structural studies of glut a m i n e PRPP amidotransferase, OPRTase, HGPRTase, and a novel UPRTase, B. subtilis PyrR (solved by D. Tomchick and J. Smith, Purdue Univ.). The enzymes share common tertiary structure and PRPP binding site, but little amino acid sequence homology outside of the PRPP site. Remarkably, this "PRTase" fold is also found and duplicated in B. subtilis PRPP synthetase (solved by T. Larsen and S. Larsen, Univ. of Copenhagen).
173 AMP DEAMINASE OF UTERINE SMOOTH MUSCLE: INFLUENCE OF ATP AND ORTHOPHOSPHATE ON AGGREGATION STATE OF THE ENZYME Szymanska, G. and Kaletha, K., Dept. Biochemistry, Med. Univ. Gdansk, 80-211 Gdansk, Poland ATP and orthophosphate are the main low molecular weight effectors which control activity of AMP deaminase by allosteric way. Some of the other enzymes of purine metabolism might be regulated additionally by the polymerization-depolymerization mechanism. Objective To check if the reversible polymerization is the putative mechanism of regulation the uterine AMP deaminase. Methods Gel filtration in the presence or absence of allosteric effectors was used. A p p a r e n t molecular weights of the active forms of AMP deaminase in the different aggregation state were estimated on Sepharose CL 6B column. Columns were preequilibrated with or without effectors and calibrated with s t a n d a r d proteins. Results Two peaks of AMP deaminase activity, one containing 280 kDa and 140 kDa molecular species and the other with 70 kDa form were detected when the purified enzyme was eluted in the absence of effectors. When ATP was present, almost 90% of total activity was found as 280 kDa oligomere. In the presence of inorganic phosphate sequence of three peaks were eluted from the column: highly polymerized (>1,000 kDa), 280 kDa and 140 kDa molecules. Conclusion Cooperating activity control for AMP deaminse of uterus smooth muscle m i g h t include two mechanisms: one allosteric limited to the enzyme subunit interactions and the other involving the reversible polymerization producing several additional active forms of the enzyme.
174 MECHANISM OF LOW SERUM URATE LEVEL IN PATIENTS WITH DIABETES MELLITUS
172 STRUCTURE A N D F U N C T I O N O F PURINE AND PYRIMIDINE METABOLIC ENZYMES
Takagi K., Inai K., Tsutani H., N a k a m u r a T., Ueda T., D e p a r t m e n t of Medicine, Fukui Medical School, Matsuoka, Fukui 910-11, J a p a n
Switzer, R.L., D e p a r t m e n t of Biochemistry, University of Illinois, 600 South Mathews, Urbana, IL 61801, USA
Objectives U r a t e metabolism in patients with diabetes mellitus
Objectives The three dimensional structures of most of the enzymes of pyrimidine nucleotide biosynthesis and of several of the 284
(DM) h a s been investigated using the u r a t e clearance test. Mean S e r u m u r a t e level (Sua) of 15 DM patients (3.83 ± 0.28 mg/dl) was significantly lower t h a n t h a t of 32 control subjects (C) CLINICAL BIOCHEMISTRY, VOLUME 30, APRIL 1997
166 APRT DEFICIENT MICE A S AN I N VIVO MAMMALIAN MODEL FOR MUTATION Peter J. Stambrook, a n d J a y A. Tischfield, University of Cincinnati, College of Medicine a n d I n d i a n a University School of Medicine There are currently no satisfactory in vivo systems for monitoring the mutagenic capacity of drugs, chemicals or environments. We are proposing the use of APRT-deficient mice carrying m u t a n t but revertible A P R T alleles as a mutagenesis model. The basis for the model is the differential capacity o f A P R T + and A P R T cells to metabolize exogenous adenine. Cells t h a t are A P R T ÷ will convert labeled adenine to labeled AMP a n d ultimately incorporate the tagged adenine derivative into nucleic acids. Cells t h a t are A P R T - will not. Objectives To determine w h e t h e r or not rare A P R T + cells (analogous to APRT + revertants) can be detected in a n A P R T background in vivo following exposure to 14C-adenine. Methods Embryonic stem (ES) cells t h a t are tumorigenic were r e n d e r e d APRT by targeted homologous recombination and subsequent selection in 2,6 diaminopurine (DAP). These APRTES cells were mixed with APRT ~ wild type ES cells at ratios of 10:1 a n d 50:1 (APRT /APRW.) a n d 5 × 106 cells were injected into each flank of a n A P R T - syngeneic mouse. Tumors were allowed to grow to 0.5-1.0 cm, at which time mice were injected I.P. with 80 ~Ci ~4C-adenine (4 ~Ci/gm wt). After 48 hours, the tumors were surgically removed, and fixed and sectioned, a n d coated with emulsion for autoradiography. Results Autoradiograms show t h a t cells in the t u m o r become labeled in proportion to the fraction o f A P R T ÷ cells in the initial innoculum. Some cells h a d nuclear and cytoplasmic labeling, indicative of DNA replication during 14C-adenine exposure. Other cells exhibited cytoplasmic labeling only. Tissues from the A P R T - host mouse lacked radioactive cells. Conclusions I.P. injection of 14C-adenine in A P R T - mice with revertible A P R T alleles will be sufficiently sensitive to detect rare A P R T + cells.
167 MODULATION OF DEOXYCYTIDINE KINASE SUBSTRATE SPECIFICITY IN LYMPHOCYTE Staub M., Keszler G., Sasv~ri-Sz6kely M. and Spasokukotskaja T., Semmelweis University of Medicine, Dept. of Med. Chem, Mol. Biol. a n d Pathobioehem., Budapest, Hungary Deoxycytidine kinase (dCK) h a s a broad s u b s t r a t e specificity, responsible to phosphorylate dCyd, dAdo, rAdo and their analogues used in therapy. The potentiation o f d C K activity h a s been shown after short t e r m t r e a t m e n t with 2-Cl-dAdo (CdA) in h u m a n tonsillar lymphocytes a n d a post-translational modification of the enzyme was suggested (1995 ESPPM, Vasta, Italy). In addition to tonsils, the "self-potentiation" by CdA was also found in h u m a n peripheral blood mononuclear cells, HL-60 cells, mouse thymocytes a n d spleen cells. The degree of stimulation was higher in G-phase, as in S-phase enriched cells. Not only CdA, but a serie of other toxic analogues can induce the potenciation of the kinase, responsible for t h e i r phosphorylation, while thymidine kinase (TK) is not affected. Even the n a t u r a l nucleoside dAdo, h a s increased the activity of dCK, if its d e a m i n a t i o n was prevented by deoxycoformycin, The concentration dependence of dAdo on the activation of dCK is biphasic, similarly to receptor binding curve of Ado, suggesting the signal transduction in the process. Inhibitors of the signal t r a n s d u c t i o n p a t h w a y were tested, a n d NaF, the universal inhibitor of phosphatases, h a s also induced the increase of dCK activity. CLINICAL BIOCHEMISTRY, VOLUME 30, APRIL 1997
The s u b s t r a t e specificity of the "potentiated" dCK was changed, induced either by the toxic analogue (CdA), or by NaF. The affinity of the enzyme to dCyd h a s been increased, while to CdA it h a s been decreased, stimulated e t h e r by CdA or by NaF. The m e c h a n i s m producing the "potentiated" nucleoside kinase might be different, induced by the nucleoside or by NaF, b u t both processes probably involve signal transduction mechanisms.
168 THIOPURINE-METHYLTRANSFERASE ACTIVITY AND TOXICITY OF AZATHIOPR1NE IN RHEUMATOID ARTHRITIS J. Stolk, A. Boerbooms, R. De Abreu*, D. de Koning*, L. v a n de P u t t e Depts. of Rheumatology & Paediatrics*, University Hospital Nijmegen P.O. Box 9101, 6500 HB Nijmegen, The N e t h e r l a n d s Objective To investigate in rheumatoid arthritis (RA) w h e t h e r purine enzyme activities may be predictive for development of azathioprine (AZA) related severe toxicity. Design & Methods P a t i e n t s with longstanding RA (n= 33) entered a prospective study for t r e a t m e n t with AZA. Before s t a r t and at m o n t h 1 and 6 of t r e a t m e n t , we m e a s u r e d activities of thiopurinem e t h y l t r a n s f e r a s e (TPMT). Controls included patients with early RA before second line t r e a t m e n t and h e a l t h y volunteers. Results Of the 33 patients 14 developed toxicity, mainly gastrointestinal intolerance, in 11 necessitating to definite withdrawal of AZA within 5 weeks after start. P a t i e n t s with toxic adverse effects h a d significantly lower TPMT activities at s t a r t and at the time of withdrawal (P <- 0.004) a n d in 50% we found a baseline intermediate TPMT activity. Of the 8 patients with baseline intermediate TPMT levels, 7 developed toxicity. There was a significant relation between intermediate TPMT activity and side effects (P = 0.005). Compared to high activity, a baseline intermediate TPMT activity resulted in a Relative Risk of 3.1 (95% CI: 1.6-6.2) to develop severe toxicity on AZA. Conclusion In RA, a p r e - t r e a t m e n t intermediate TPMT activity is predictive for development of severe toxicity on AZA. As clinical implication one should consider TPMT m e a s u r e m e n t prior to treatment. Gastrointestinal side effects are not necessarily caused by 'hypersensitivity', but may be based on a (thio)purine metabolic disbalance.
169 ARE SUCCINYLPURINES NEUROTOXIC? Stone, T.W., Roberts, L.A., Morris, B.J., Jones, P.A., Duley, J.A. and Ogilvy, H.V., West Medical Building, University of Glasgow, Glasgow G12 8QQ, Scotland Objectives P a t i e n t s with a deficiency of adenylosuccinate lyase exhibit m e n t a l retardation, a u t i s m a n d epilepsy, with a n accumulation of two abnormal purines succinyladenosine (S-aden) and succinylaminoimidazole carboxamide ribotide (SAICAr) in the CSF. We have now tried to determine w h e t h e r these could produce neurotoxic effects in the brain. Design and Methods Each of the purines was applied initially by microinjection directly into the hippocampus of anaesthetised rats, and in later experiments by reverse microdialyses over a period of 10 hours. The solution injected or dialysed contained 2 mmolesfl of the abnormal purines. In b o t h cases, the animals were allowed to recover for 7 days, w h e n the b r a i n s were removed for histological analysis. Results The initial, single injections of each purine did not induce any signs of neuronal damage or degeneration, perhaps because the compounds were not present for a sufficiently long period. However, after reverse microdialysis, 5 of 6 animals receiving
283
9TH INTERNATIONAL/6TH EUROPEAN J O I N T SYMPOSIUM ON PURINE AND PYRIMIDINE METABOLISM IN MAN SAICAr showed significant degeneration of pyramidal neurones. S-aden h a d no effect. Conclusion SAICAr h a s a neurotoxic action on pyramidal neurones of the r a t hippocampus. If a similar action occurs elsewhere, and in h u m a n s , such neuronal damage could contribute to the m e n t a l retardation, a u t i s m a n d epilepsy found in some p a t i e n t s with adenylosuccinase deficiency.
170 SUCCINYLPURINES DO NOT MODIFY GLUTAMATE OR A D E N O S I N E EFFECTS IN THE CNS Stone, T.W., De Abreu, R.A., Duley, J.A., Gross, M., Salerno, C., v a n den Berghe, G.
Objectives To determine if the abnormal purines detected in patients suffering from deficiency of adenylosuccinate lyase, succinyladenosine (S-aden) a n d succinylaminoimidazole carboxamide ribotide (SAICAr) have any effect on synaptic transmission in the hippocampus. Design and Methods Slices 500 p,m thick were prepared from male rats a n d prepared for recordings of synaptic potentials evoked from the s t r a t u m r a d i a t u m a n d single cell firing rates. Results S-aden a n d SAICAr did not modify either the evoked population spike potential or the evoked population excitatory postsynaptic potential (glutamatergic) at concentrations up to 0.2 mM, the concentration found in the CSF of patients. These purines did not modify responses of single cells to g l u t a m a t e or several analogues. Adenosine itself depressed synaptic potentials, in the range of 5-50 ~LM. However S-aden or SAICAr did not modify, by blocking or enhancing, the effects of adenosine. Conclusion Neither of the abnormal purines modify synaptic transmission in the r a t hippocampus. It seems unlikely t h a t a disruption of synaptic transmission contributes substantially to the CNS symptoms of some p a t i e n t s with adenylosuccinase deficiency.
171 URINARY SCREENING FOR PYRIMIDINE METABOLISM DISORDERS: REFERENCE RANGES FOR DIHYDROURACIL, URACIL A N D DIHYDROURACI/URACIL RATIO Satoshi Sumi, Kiyoshi Kidouchi, Katsuo Hayashi, Masaynki Imaeda, Masami Asai, Yoshiro Wada
Objectives Pyrimidine chemotherapy agent such as 5-FU are used widely but can occasionally cause serious adverse reactions in patients with pyrimidine metabolism disorders. We have analyzed u r i n a r y pyrimidine levels in 167 h e a l t h y adults and 966 p a t i e n t s with malignancy, hypertension, cerebral infarction, etc. The reference range of dihydrouracil for h e a l t h y adults was 23.8 ± 35.5 (mean ± S.D.; ~mol/g creatinine) and t h a t of uracil was 63.3 -+ 66.0. T h a t ofdihydrouracil/uracil ratio was 0.31 ± 0.16. In addition, a n asymptomatic m a n with dihydropyrimidinuria was detected in this study. We performed a n oral uracil loading test on him. The uracil and the dihydrouracil level in his blood were extremely high after the test, suggesting t h a t the 5-FU level would be also high if it was treated.
enzymes of purine nucleotide biosynthesis have been determined or are currently being determined at high resolution by x-ray diffraction of crystals. The structures of a n u m b e r of enzymes of nucleobase salvage and catabolism have also been solved. These structures provide a wealth of information about mechanisms of catalysis, consequences of deleterious mutations in humans, and potential strategies for drug development. Valuable generalizations concerning enzymatic mechanisms of several reaction types t h a t are i m p o r t a n t in nucleotide metabolism, such as phosphoribosyltransfer, glutamine dependent amination, and a s p a r t a t e dependent amination, have emerged from the structural studies. These concepts will be illustrated with a n analysis of phosphoribosyltransferase enzymes based on structural studies of glut a m i n e PRPP amidotransferase, OPRTase, HGPRTase, and a novel UPRTase, B. subtilis PyrR (solved by D. Tomchick and J. Smith, Purdue Univ.). The enzymes share common tertiary structure and PRPP binding site, but little amino acid sequence homology outside of the PRPP site. Remarkably, this "PRTase" fold is also found and duplicated in B. subtilis PRPP synthetase (solved by T. Larsen and S. Larsen, Univ. of Copenhagen).
173 AMP DEAMINASE OF UTERINE SMOOTH MUSCLE: INFLUENCE OF ATP AND ORTHOPHOSPHATE ON AGGREGATION STATE OF THE ENZYME Szymanska, G. and Kaletha, K., Dept. Biochemistry, Med. Univ. Gdansk, 80-211 Gdansk, Poland ATP and orthophosphate are the main low molecular weight effectors which control activity of AMP deaminase by allosteric way. Some of the other enzymes of purine metabolism might be regulated additionally by the polymerization-depolymerization mechanism. Objective To check if the reversible polymerization is the putative mechanism of regulation the uterine AMP deaminase. Methods Gel filtration in the presence or absence of allosteric effectors was used. A p p a r e n t molecular weights of the active forms of AMP deaminase in the different aggregation state were estimated on Sepharose CL 6B column. Columns were preequilibrated with or without effectors and calibrated with s t a n d a r d proteins. Results Two peaks of AMP deaminase activity, one containing 280 kDa and 140 kDa molecular species and the other with 70 kDa form were detected when the purified enzyme was eluted in the absence of effectors. When ATP was present, almost 90% of total activity was found as 280 kDa oligomere. In the presence of inorganic phosphate sequence of three peaks were eluted from the column: highly polymerized (>1,000 kDa), 280 kDa and 140 kDa molecules. Conclusion Cooperating activity control for AMP deaminse of uterus smooth muscle m i g h t include two mechanisms: one allosteric limited to the enzyme subunit interactions and the other involving the reversible polymerization producing several additional active forms of the enzyme.
174 MECHANISM OF LOW SERUM URATE LEVEL IN PATIENTS WITH DIABETES MELLITUS
172 STRUCTURE A N D F U N C T I O N O F PURINE AND PYRIMIDINE METABOLIC ENZYMES
Takagi K., Inai K., Tsutani H., N a k a m u r a T., Ueda T., D e p a r t m e n t of Medicine, Fukui Medical School, Matsuoka, Fukui 910-11, J a p a n
Switzer, R.L., D e p a r t m e n t of Biochemistry, University of Illinois, 600 South Mathews, Urbana, IL 61801, USA
Objectives U r a t e metabolism in patients with diabetes mellitus
Objectives The three dimensional structures of most of the enzymes of pyrimidine nucleotide biosynthesis and of several of the 284
(DM) h a s been investigated using the u r a t e clearance test. Mean S e r u m u r a t e level (Sua) of 15 DM patients (3.83 ± 0.28 mg/dl) was significantly lower t h a n t h a t of 32 control subjects (C) CLINICAL BIOCHEMISTRY, VOLUME 30, APRIL 1997