- Email: [email protected]
Effect of FMLP stimulation on [3H]-NECA binding to adenosine receptors in neutrophils membranes
POSTER ABSTRACTS then loaded on a PBE94 column developed using increasing NaC1 concentrations. Results ADA, AK, AMP deaminase, 5'-N, PNP, ADPase and ATPase were completely removed while MK activity was still contaminant. Conclusion We obtained a partially purified AMP-AMP PT preparation with a specific activity of about 1000 nmoles/iffmg protein.
quinone binding site and provided further evidence for structural zinc. Conclusions These findings could assist inhibitor-structure-activity relation studies with considerable understanding. [1] W. Knecht, U. Bergjohann, S. Gonski, B. Kirschbaum, M. Lbffier (1996) Eur. J. Biochem. 240, 292-301. [2] W. Knecht, D. Altekruse, A. Rotgeri, S. Gonski, M. LOftier (1997) Prot. Expr. Purif., in press.
99
101 EFFECT OF FMLP STIMULATION ON [3H]-NECA BINDING TO ADENOSINE RECEPTORS IN NEUTROPHILS MEMBRANES
MEASUREMENT OF MUTATION FREQUENCY AT THE HPRT LOCUS IN PERIPHERAL LYMPHOCYTES; IS THIS A GOOD METHOD TO EVALUATE A CANCER RISK IN PEDIATRIC PATIENTS? Ying-Wei Lin, Masaru Kubota, Machiko Sawada, Yuichi Akiyama and Kenshi Furusho, Department of Pediatrics, Kyoto University, Kyoto 606, Japan
Objectives Validity of measurement of somatic cell mutation frequency (MD at HPRT locus for evaluating cancer risk of the given individual was determined in pediatric patients. Design and Methods Peripheral lymphocytes (PL) from patients with various diseases, including malignancies (before or after chemotherapy), DNA-repair deficient syndromes or short statue receiving growth hormone (GH), were isolated through FicollHypaque sedimentation with informed consent. Mf at the HPRT locus of PL was determined by limiting dilution assay using 6-thioguanine. Results (1) ALL patients after chemotherapy had higher Mf than that of age-matched controls. (2) Patients with Hodgkin's disease or some solid tumor tended to have higher Mf even before chemotherapy. (3) Among DNA-repair deficient syndromes, diseases which are susceptible to cancer (XP, AT) have high Mf, but those without any cancer disposition (Cockayne syndrome) have normal Mf. (4) GH-receiving patients have normal Mf, regardless of total doses of GH. Conclusion Measurement of Mf a: HPRT locus may be useful for evaluating cancer risk of pediatric patients.
100 DIHYDROOROTATE DEHYDROGENASE: PROFILE OF A NOVEL TARGET FOR ANTIPROLIFERATIVE AND IMMUNOSUPPRESSIVE DRUGS Monika LSffier, Institute for Physiological Chemistry, Karlvon-Frisch-Str. 1 D-35033 Marburg, Germany Whilst the structure and thnction of cytosolic enzymes--the multifunctional CAD and UMPsynthase--are quite well understood, the membrane-bound mitochondrial and low-abundant tissue DHOdehases have evaded biochemical elucidation. No inborn errors have been reported. Objectives Recent attention has focussed on DHOdehase following its identification as the target of drugs effective against autoimmune diseases and reactions leading to graft rejection. These studies and associated clinical trials restimulated the discussion on the importance of pyrimidine nucleotides for immunocompetence, and te what extent the separate location of DHOdehase in the mitochondria could be advantageous for intervention in pool adjustment. Methods and Results The over-expression of the human and rodent enzymes in a heterologous system [1,2] provided plentiful and homogeneous recombinant material for enzyme investigation in vitro. The availability of anti-DHOdehase IgG here enabled cell and tissue studies. As discussed in detail in separate presentations (this symposium), recent data identified the flavin FMN as the main redox center, excluded iron-sulfur-centers, confirmed a CLINICAL BIOCHEMISTRY, VOLUME 30, APRIL 1997
Martini C., Trincavelli L., Fiorini M., Nardi M., Bazzichi L.*, Lucacchini A. Istituto Policattedra di Discipline Biologiche. *Istituto Patologia Speciale Medica, University of Pisa, Pisa, Italy
Objectives [3H]-NECA binding to adenosine receptors were characterized in FMLP stimulated polymorphonuclear leucocyte (PMN) membranes vs control. Design and Methods FMLP (formyl methionyl leucil phenil alanine) and adenosine receptors are coexisting in PMN membranes. FMLP binding to its specific receptors sites stimulates superoxide anion generation, aggregation and degranulatien; adenosine binding to A1 receptors promotes chemotaxis, whereas stimulation of A2 receptors inhibits superoxide anion generation. Membranes obtained by 50 ~M FMLP stimulated neutrophils were incubated with 13 nM [3H]-NECA in the presence and in the absence of 100 vLM NECA or R-PIA to determine non specific binding. 50 nM CPA was used to block [3H]-NECA binding to A1 adenosine receptors. Saturation experiments are carried out with increasing [3H]-NECA concentrations. Results In the presence of the stimulus the mean value of Kd and Bmax was 510 nM and 2661 fmol/mg respectively, whereas in absence ef stimulus the Kd values was 883 nM and Bmax was 5187 fmol/mg. Conclusion Several studies are in progress to determine the contibute of the two distinct NECA binding sites (A2 and A2-1ike) on this binding characteristics modulation.
102 MAXIMIZING RESIDUAL AMP DEAMINASE EXPRESSION IN MYOADENYLATE DEAMINASE DEFICIENCY THROUGH THE FORMATION OF HETEROTETRAMERS OF ISOFORMS M AND E D.K. Mahnke-Zizelman and R.L. Sabina, Medical College of Wisconsin, Milwaukee, WI, USA
Objective To examine the potential impact of isoform E expression in myoadenylate deaminase (isoform M) "deficient" skeletal muscle. Design and Methods Recombinant human AMPD1 (isoferm M) and AMPD3 (isoform E) cDNAs were co-expressed from a baculoviral vector to determine whether M]E heterotetrameric enzymes could be synthesized. Resultant hybrid activities were partially purified and characterized. Results Multiple M]E activities were resolved by phosphocellulose chromatography and shown to exhibit features similar to isoform M, including activation by ATP, phosphorylation by protein kinase C and affinity for actinomyosin. Conclusion The production of M/E hybrid activities capable of responding to physiological regulators of isoform M may enhance the catalytic potential for residual enzyme expression in myoadenylate deaminase "deficient" skeletal muscle resulting from alternative splicing of the AMPD1 primary transcript. 267
quinone binding site and provided further evidence for structural zinc. Conclusions These findings could assist inhibitor-structure-activity relation studies with considerable understanding. [1] W. Knecht, U. Bergjohann, S. Gonski, B. Kirschbaum, M. Lbffier (1996) Eur. J. Biochem. 240, 292-301. [2] W. Knecht, D. Altekruse, A. Rotgeri, S. Gonski, M. LOftier (1997) Prot. Expr. Purif., in press.
99
101 EFFECT OF FMLP STIMULATION ON [3H]-NECA BINDING TO ADENOSINE RECEPTORS IN NEUTROPHILS MEMBRANES
MEASUREMENT OF MUTATION FREQUENCY AT THE HPRT LOCUS IN PERIPHERAL LYMPHOCYTES; IS THIS A GOOD METHOD TO EVALUATE A CANCER RISK IN PEDIATRIC PATIENTS? Ying-Wei Lin, Masaru Kubota, Machiko Sawada, Yuichi Akiyama and Kenshi Furusho, Department of Pediatrics, Kyoto University, Kyoto 606, Japan
Objectives Validity of measurement of somatic cell mutation frequency (MD at HPRT locus for evaluating cancer risk of the given individual was determined in pediatric patients. Design and Methods Peripheral lymphocytes (PL) from patients with various diseases, including malignancies (before or after chemotherapy), DNA-repair deficient syndromes or short statue receiving growth hormone (GH), were isolated through FicollHypaque sedimentation with informed consent. Mf at the HPRT locus of PL was determined by limiting dilution assay using 6-thioguanine. Results (1) ALL patients after chemotherapy had higher Mf than that of age-matched controls. (2) Patients with Hodgkin's disease or some solid tumor tended to have higher Mf even before chemotherapy. (3) Among DNA-repair deficient syndromes, diseases which are susceptible to cancer (XP, AT) have high Mf, but those without any cancer disposition (Cockayne syndrome) have normal Mf. (4) GH-receiving patients have normal Mf, regardless of total doses of GH. Conclusion Measurement of Mf a: HPRT locus may be useful for evaluating cancer risk of pediatric patients.
100 DIHYDROOROTATE DEHYDROGENASE: PROFILE OF A NOVEL TARGET FOR ANTIPROLIFERATIVE AND IMMUNOSUPPRESSIVE DRUGS Monika LSffier, Institute for Physiological Chemistry, Karlvon-Frisch-Str. 1 D-35033 Marburg, Germany Whilst the structure and thnction of cytosolic enzymes--the multifunctional CAD and UMPsynthase--are quite well understood, the membrane-bound mitochondrial and low-abundant tissue DHOdehases have evaded biochemical elucidation. No inborn errors have been reported. Objectives Recent attention has focussed on DHOdehase following its identification as the target of drugs effective against autoimmune diseases and reactions leading to graft rejection. These studies and associated clinical trials restimulated the discussion on the importance of pyrimidine nucleotides for immunocompetence, and te what extent the separate location of DHOdehase in the mitochondria could be advantageous for intervention in pool adjustment. Methods and Results The over-expression of the human and rodent enzymes in a heterologous system [1,2] provided plentiful and homogeneous recombinant material for enzyme investigation in vitro. The availability of anti-DHOdehase IgG here enabled cell and tissue studies. As discussed in detail in separate presentations (this symposium), recent data identified the flavin FMN as the main redox center, excluded iron-sulfur-centers, confirmed a CLINICAL BIOCHEMISTRY, VOLUME 30, APRIL 1997
Martini C., Trincavelli L., Fiorini M., Nardi M., Bazzichi L.*, Lucacchini A. Istituto Policattedra di Discipline Biologiche. *Istituto Patologia Speciale Medica, University of Pisa, Pisa, Italy
Objectives [3H]-NECA binding to adenosine receptors were characterized in FMLP stimulated polymorphonuclear leucocyte (PMN) membranes vs control. Design and Methods FMLP (formyl methionyl leucil phenil alanine) and adenosine receptors are coexisting in PMN membranes. FMLP binding to its specific receptors sites stimulates superoxide anion generation, aggregation and degranulatien; adenosine binding to A1 receptors promotes chemotaxis, whereas stimulation of A2 receptors inhibits superoxide anion generation. Membranes obtained by 50 ~M FMLP stimulated neutrophils were incubated with 13 nM [3H]-NECA in the presence and in the absence of 100 vLM NECA or R-PIA to determine non specific binding. 50 nM CPA was used to block [3H]-NECA binding to A1 adenosine receptors. Saturation experiments are carried out with increasing [3H]-NECA concentrations. Results In the presence of the stimulus the mean value of Kd and Bmax was 510 nM and 2661 fmol/mg respectively, whereas in absence ef stimulus the Kd values was 883 nM and Bmax was 5187 fmol/mg. Conclusion Several studies are in progress to determine the contibute of the two distinct NECA binding sites (A2 and A2-1ike) on this binding characteristics modulation.
102 MAXIMIZING RESIDUAL AMP DEAMINASE EXPRESSION IN MYOADENYLATE DEAMINASE DEFICIENCY THROUGH THE FORMATION OF HETEROTETRAMERS OF ISOFORMS M AND E D.K. Mahnke-Zizelman and R.L. Sabina, Medical College of Wisconsin, Milwaukee, WI, USA
Objective To examine the potential impact of isoform E expression in myoadenylate deaminase (isoform M) "deficient" skeletal muscle. Design and Methods Recombinant human AMPD1 (isoferm M) and AMPD3 (isoform E) cDNAs were co-expressed from a baculoviral vector to determine whether M]E heterotetrameric enzymes could be synthesized. Resultant hybrid activities were partially purified and characterized. Results Multiple M]E activities were resolved by phosphocellulose chromatography and shown to exhibit features similar to isoform M, including activation by ATP, phosphorylation by protein kinase C and affinity for actinomyosin. Conclusion The production of M/E hybrid activities capable of responding to physiological regulators of isoform M may enhance the catalytic potential for residual enzyme expression in myoadenylate deaminase "deficient" skeletal muscle resulting from alternative splicing of the AMPD1 primary transcript. 267