Demonstration of induction of erythrocyte inosine monophosphate dehydrogenase activity in Ribavirin-treated patients using a high performance liquid chromatography linked method.
ABSTRACT The activity of inosine monophosphate dehydrogenase (IMPDH: EC 22.214.171.124) was measured in erythrocyte lysates using a non-radiolabelled method linked to reversed-phase liquid chromatography (RPLC). The mean activity in erythrocytes from healthy controls using this sensitive method was extremely low (mean 85 pmol/h per mg protein, range 4-183). The elevated erythrocyte IMPDH activity reported previously in hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency was confirmed (mean 234 pmol/h per mg protein). Erythrocyte IMPDH activity of patients with other disorders of purine metabolism, or with leukaemias and lymphomas, showed no marked difference from controls, except in one instance--an immunodeficient child with purine nucleoside phosphorylase (PNP) deficiency, treated with Ribavirin, where a 30-fold increase in activity was found (2670 pmol/h per mg protein). Investigation of erythrocyte IMPDH in other immunodeficient children with normal PNP activity demonstrated that this grossly elevated erythrocyte activity was attributable to induction of IMPDH by Ribavirin therapy.
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ABSTRACT: The nucleotide concentrations in acid and alkaline erythrocyte extracts have been measured by RP-HPLC in healthy controls and in patients bearing different inherited disorders, with altered erythrocyte NAD(P) levels. The objective was the simultaneous determination of the nucleotide profile and of the oxidative state of pyridine coenzymes by the most suitable extraction method. Both alkaline and acid extractions were necessary to obtain the complete pattern, due to defective recovery of the oxidized or reduced coenzymes, respectively, during the extraction procedures. Purine nucleotide quantification seemed to be reliable by all methods. High NADP+ levels were confirmed in two glucose-6-phosphate dehydrogenase deficient patients, coupled with raised NAD levels, lowered NADPH/NADP+ ratio and increased NADH/NAD+ ratio. Higher NAD+ and normal or lower NADH/NAD+ ratios were found in two hypoxanthine-phosphoribosyltransferase deficient patients, while a patient with superactive phosphoribosylpyrophosphate synthetase showed a decreased NADH level in addition to the low NAD+ level previously found.Clinica Chimica Acta 11/1993; 220(1):1-17. · 2.85 Impact Factor
Article: Regulation of GTP biosynthesis.[show abstract] [hide abstract]
ABSTRACT: In the regulation of GTP biosynthesis, complex interactions are observed. A major factor is the behavior of the activity of IMPDH, the rate-limiting enzyme of de novo GTP biosynthesis, and the activity of GPRT, the salvage enzyme of guanylate production. The activities of GMP synthase, GMP kinase and nucleoside-diphosphate kinase are also relevant. In neoplastic transformation, the activities and amounts of all these biosynthetic enzymes are elevated as shown by kinetic assays and by immunotitration for IMPDH. In cancer cells, the up-regulation of guanylate biosynthesis is amplified by the concurrent decrease in activities of the catabolic enzymes, nucleotidase, nucleoside phosphorylase, and the rate-limiting purine catabolic enzyme, xanthine oxidase. The up-regulation of the capacity for GTP biosynthesis is also manifested in the stepped-up capacity of the overall pathways of de novo and salvage guanylate production. The linking with neoplasia is also seen in the elevation of the activities of IMPDH and GMP synthase and de novo and salvage pathways as the proliferative program is expressed as cancer cells enter log phase in tissue culture. The activity of GMP reductase showed no linkage with neoplastic or normal cell proliferation; however, in induced differentiation in HL-60 cells the activity increased concurrently with the decline in the activity of IMPDH. This reciprocal regulation of the two enzymes is observed in differentiation induced by retinoic acid, DMSO or TPA in HL-60 cells. In support of enzyme-pattern-targeted chemotherapy, evidence was provided for synergistic chemotherapy with tiazofurin (inhibitor of IMPDH) and hypoxanthine (competitive inhibitor of GPRT and guanine salvage activity) in patients and in tissue culture cell lines. These investigations should contribute to the clarification of the controlling factors of GMP biosynthesis, the role of the various enzymes, the behavior of GMP reductase in mammalian cells and the application of the approaches of enzyme-pattern-targeted chemotherapy in patients.Advances in Enzyme Regulation 02/1992; 32:57-69.
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ABSTRACT: Mouse neuroblastoma cells (NB) selected for 10,000-fold increased resistance to mycophenolic acid (NB-Myco) showed a 200-500-fold increase in IMP dehydrogenase protein, and the enzyme (IMP: NAD+ oxidoreductase, EC 126.96.36.199) also exhibited a 2400-fold increased ki for mycophenolic acid and reduced catalytic efficiency (Hodges, S.D., Fung, E., McKay, D.J., Renaux, B.S., and Snyder, F.F. (1989) J. Biol. Chem. 264, 18137-18141). The molecular basis of these changes is the subject of the present study. The nucleotide sequence of IMP dehydrogenase cDNA from NB-Myco cells revealed four nucleotide changes. One of these changes did not result in a codon change, and a second one corresponding to methionine-483 was present in the parental NB mouse line. The remaining two nucleotide substitutions and deduced residue changes are: the C to T transition at base 998 relative to initiation of translation, altering threonine-333 to isoleucine; and the C to A transversion at base 1052, altering serine-351 to tyrosine. Evidence was also obtained for IMP dehydrogenase having undergone gene amplification. IMP dehydrogenase mRNA levels were 500-fold increased in NB-Myco cells as compared to parental NB cells. DNA dot blot analysis showed a 25-fold increase in IMP dehydrogenase gene copy number and restriction enzyme analysis revealed similar gene structure for NB and NB-myco cells.Biochimica et Biophysica Acta 04/1994; 1217(2):156-62. · 4.66 Impact Factor