Adenine-induced hypoxanthine release from IMP-enriched human erythrocytes

Adenine uptake and hypoxanthine release by IMP-enriched human erythrocytes has been studied. The presence of IMP within the erythrocytes leads to an increase in the rate of adenine incorporation. Adenine is taken up by IMP-enriched erythrocytes as AMP, even when intracellular 5-phoshorobosyl-1-pyrophosphate concentration is undetectable and too low to allow IMP synthesis from hypoxanthine. During adenine uptake and AMP synthesis, hypoxanthine is released by the cells. The possibility that 5-phosphoribosyl-1-pyrophosphate, necessary for AMP synthesis, is formed through the hypoxanthine guanine phosphoribosyltransferese-catalyzed IMP pyrophosphorolysis is considered.     

Human hypoxanthine-guanine phosphoribosyltransferase. IMP-GMP exchange stoichiometry and steady state kinetics of the reaction.

Steady state kinetics of hypoxanthine guanine phosphoribosyltransferase-catalyzed reactions are studied. The results obtained suggest that IMP, GMP, P-Rib-PP, and pyrophosphate bind to the same enzyme form, while hypoxanthine and guanine bind to a different form. Guanine activates IMP-pyrophosphorolysis. During the reaction guanine and IMP are consumed with formation of GM     

Hypoxanthine-guanine exchange by intact human erythrocytes

The uptake and release of [14C]hypoxanthine by human erythrocytes, suspended in a tris(hydroxymethyl)aminomethane (Tris)-glucose-NaCl isotonic medium (pH 7.4), have been studied at 37 degrees C. The uptake of hypoxanthine, mediated by its incorporation into inosine 5'-monophosphate (IMP), was markedly stimulated by preincubating the cells in phosphate-buffered saline. After a lag time, [14C]IMP-enriched erythrocytes released [14C]hypoxanthine in the medium. Formycin B, at concentrations known to inhibit purine nucleoside phosphorylase in intact erythrocytes, affected hypoxanthine uptake and release and led to an increase in the intracellular concentration of inosine, suggesting that the main catabolic path of IMP is the sequential degradation of the nucleotide to inosine and hypoxanthine. The addition of guanine to a suspension of [14C]IMP-enriched erythrocytes led to an increase in the rate of [14C]hypoxanthine release, which was unaffected by the presence of formycin B. During the guanine-induced hypoxanthine release, guanine was taken up by the cells as GMP. These results suggest that the presence of guanine in the incubation medium activates a catabolic path in human erythrocytes leading to IMP degradation without formation of inosine.     

The biochemical determinants of hypoxanthine uptake in Novikoff rat hepatoma cells

The rate with which Novikoff rat hepatoma cells took up exogenous hypoxanthine increased sharply towards the end of the logarithmic growth phase, remained high for several hours into the stationary phase, and then decreased again. In an effort to account for these phenomena, several biochemical parameters were monitored during culture growth: the activities of the hypoxanthine transporter, of hypoxanthine phosphoribosyltransferase, and of P-Rib-PP synthetase; and the intracellular concentrations of ATP and P-Rib-PP. All of these parameters remained virtually constant during growth of the culture, except for P-Rib-PP, which increased greater than 10-fold in a pattern similar to that for hypoxanthine uptake. The activities of the transporter, synthetase, and phosphoribosyltransferase remained stable over 7 h of treatment with cycloheximide.     

Human hypoxanthine guanine phosphoribosyltransferase. The role of magnesium ion in a phosphoribosylpyrophosphate-utilizing enzyme

The role of Mg ions in the hypoxanthine guanine phosphoribosyltransferase-catalyzed reaction have been studied using accurate values of proton and Mg stability constants of phosphoribosylpyrophosphate (P-Rib-PP) determined from pH titration data. The results obtained favor the conclusion that the dimagnesium salt of P-Rib-PP is the true substrate of the enzyme. The other species of P-Rib-PP do not appreciably affect the initial reaction rate. The inhibition of the hypoxanthine guanine phosphoribosyltransferase-catalyzed reaction observed at high MgCl2 concentration can be attributed to a competitive inhibition of Mg2+ with respect to the dimagnesium salt of P-Rib-PP, suggesting that these ionic species bind to the same enzyme form. At a fixed [P-Rib-PPtot], the concentration of its dimagnesium complex is a sigmoidal function of MgCl2 concentration, suggesting that caution must be employed in the interpretation of sigmoidal saturation curves for P-Rib-PP-utilizing enzymes when low and not constant concentrations of the divalent cation are used.     

Partial hypoxanthine-guanine phosphoribosyl transferase deficiency with full expression of the Lesch-Nyhan syndrome

Summary A patient with the full clinical expression of the classical Lesch-Nyhan syndrome is presented with a residual hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity of 5–10% in erythrocyte lysate and about 30% in fibroblast lysate. The activities of other erythrocyte enzymes of purine metabolism were typical for a classical Lesch-Nyhan patient. The effects of allopurinol therapy on the excretion of urinary purine metabolites were studied by a newly developed isotachophoretic technique.The unusually high residual activity of HGPRT in erythrodytes and fibroblasts of the patient enabled the enzymologic characterization of the mutant enzyme: in fibroblasts the affinities for the substrates hypoxanthine and guanine were normal. However, there was an increased apparent K _m for phosphoribosylpyrophosphate (PRPP), a complete absence of product inhibition by IMP and GMP, and a decreased heat stability. Addition of PRPP did not stabilize the mutant enzyme. In addition to the altered properties of the fibroblast enzyme, the K _m of the erythrocyte enzyme for hypoxanthine was also increased.Immunoprecipitation experiments revealed the presence of an approximately normal amount of material cross-reacting with anti-human HGPRT antiserum. However, it appeared that this cross-reacting material had a decreased stability. When intact erythrocytes were incubated with radiolabeled purine bases, no formation of IMP or GMP could be detected, despite the relatively high residual activity of HGPRT in the hemolysate. The results fit the following hypothesis: as a consequence of a structural mutation affecting the PRPP-site of the enzyme and a decreased heat stability, the activity of the mutant enzyme under in vivo conditions is virtually zero.In the erythrocytes of the patient's mother a normal HGPRT-activity was found. However, the activity in her fibroblasts was lower than normal, while a decreased heat stability and an intermediate behavior towards IMP could be shown.Hair root analysis of several members of the patient's family confirmed the heterozygosity of the mother, whereas no other heterozygotes could be detected. The family anamnesis did not show other cases of Lesch-Nyhan syndrome. These findings were taken as evidence that the patient described in this paper might represent a mutation orginating from the gametes in either of the maternal grandparents.     

Regulation of purine de novo synthesis in cultured human fibroblasts: the role of P-ribose-PP.

Procedures for assaying the rate of purine de novo synthesis in cultured fibroblast cells have been compared. These were (i) the incorporation of [(14)C]-glycine or [(14)C]formate in alpha-N-formylglycinamide ribonucleotide (an intermediate in the purine synthetic pathway) and (ii) the incorporation of [(14)C]-formate into newly synthesised cellular purines and purines excreted by the cell into the medium. Fibroblast cells, derived from patients with a deficiency of hypoxanthine phosphoribosyltransferase (HPRT-) (EC 2.4.2.8) and increased rates of purine de novo synthesis, were compared with fibroblasts from healthy subjects (HPRT+). Fetal calf serum, which was used to supplement the assay and cell growth medium, was found to contain sufficient quantities of the purine base hypoxanthine to inhibit purine de novo synthesis in HPRT+ cells. This inhibition was the basis of differentiation between HPRT- and HPRT+ cells. In the absence of added purine base, both cell types had similar capacities for purine de novo synthesis. This result contrasts with the increased rates of purine de novo synthesis reported for a number of human HPRT- cells in culture but conforms recent studies made on human HPRT- lymphoblast cells. The intracellular concentration and utilisation of 5-phosphoribosyl-1-pyrophosphate (P-Rib-PP), a substrate and potential controlling factor for purine de novo synthesis, were determined in HPRT- and HPRT+ cells. The rate of utilisation of P-Rib-PP in the salvage of free purine bases was far greater than that in purine de novo synthesis. Although HPRT- cells had a 3-fold increase in P-Rib-PP content, the rate of P-Rib-PP generation was similar to HPRT+ cells. Thus, in fibroblasts, the concentration of P-Rib-PP appears to be critical in the control of de novo purine synthesis and its preferential utilisation in the HPRT reaction limits its availability for purine de novo synthesis. In vivo, HPRT+ cells, in contrast to HPRT- cells, may be operating purine de novo synthesis at a reduced rate because of their ability to reutilise hypoxanthine.     

The separation of adenine and hypoxanthine-guanine phosphoribosyl transferases isoenzymes by disc gel electrophoresis

Hypoxanthine-guanine (HGPRT; E.C. 2.4.2.8) and adenine (APRT; E.C. 2.4.2.7) phosphoribosyl transferases were studied by disc electrophoresis on polyacrylamide gel. The positions of the isoenzymes were detected by radiochemical enzyme assay. The nucleotide products of the reactions were precipitated in the gel with lanthanum chloride. APRT was found to migrate slightly less rapidly than albumin and produced a single narrow symmetrical peak of activity. HGPRT migrated 25–50% more slowly than albumin and produced a broad zone of activity consisting of four unequal peaks. The APRT enzyme of Rhesus monkey liver and the HGPRT enzyme of sheep erythrocytes migrated notably slower than the corresponding human enzymes. An isoenzyme of APRT was detected in human erythrocytes which migrated more rapidly than that of most individuals. In all instances, the adenine was utilized by one electrophoretic component and hypoxanthine and guanine by another. Furthermore, the components which utilized hypoxanthine and guanine were inseparable. The sensitivity of the assay made it possible to assess the electrophoretic and enzymatic characteristics of HGPRT isoenzymes on aliquots of hemolysates capable of producing 0.5 picomoles of IMP per minute. In human erythrocytes with normal enzyme content, this amount of activity is present in approximately 50 nanoliters of cells.Aided by U.S. Public Health Service grants Nos. HD 04608 and HD 03015 from the National Institute of Child Health and Human Development, National Institutes of Health.     

Studies on IMP degradation and ribose 1-phosphate utilization in human erythrocytes

1. Intact human red cells do not attack exogenous IMP. The nucleotide is readily broken down by the soluble erythrocyte fraction to inosine, hypoxanthine and ribose 1-phosphate, with a pH optimum of approx. 6.2. 2. Ribose 1-phosphate can be actively reutilized, in the presence of ATP and hypoxanthine, to give IMP, at pH 7.4. The velocity of the IMP salvage synthesis dramatically increases at more alkaline pH values. 3. The two curves relating the velocities of IMP breakdown and of IMP synthesis as a function of hydrogen ion concentration intersect at pH 7.4. 4. The observations might be relevant in the process of purine transport by red cells.     

Nature of 6-methylpurine inhibition and characterization of two 6-methylpurine-resistant mutants of Neurospora crassa.

6-Methylpurine, an analog of adenine, inhibits the growth of Neurospora crassa. From kinetic studies it was found that 6-methylpurine is converted to its nucleotide form by adenine phosphoribosyltransferase (EC 2.4.2.7), and inhibits the de novo purine biosynthesis. Adenine relieves the growth inhibition caused by 6-methylpurine, whereas hypoxanthine is not very effective. Studies dealing with hypoxanthine utilization in the presence of 6-methylpurine indicated a severely reduced uptake of hypoxanthine and a general slowdown in its further metabolism. Two mutants (Mepr-3 and Mepr-10) which are resistant to 6-methylpurine were characterized. Studies of purine base uptake and the in vivo and in vitro conversion to nucleotides indicated that Mepr-10 may be an adenine phosphoribosyltransferase-defective mutant, whereas Mepr-3 may be a mutant with altered feedback response to 6-methylpurine. Both mutants showed a severely lowered hypoxanthine phosphoribosyltransferase activity, but because 6-methylpurine did not have any effect on the conversion of hypoxanthine to IMP in the wild type, it was concluded that 6-methylpurine resistance in these mutants cannot be due to lowered hypoxanthine phosphoribosyltransferase activity, but rather that the lowering of enzyme activity may be a secondary effect.     

High Km soluble 5'-nucleotidase from human placenta. Properties and allosteric regulation by IMP and ATP

A human placental soluble "high Km" 5'-nucleotidase has been separated from "low Km" 5'-nucleotidase and nonspecific phosphatase by AMP-Sepharose affinity chromatography. The enzyme was purified 8000-fold to a specific activity of 25.6 mumol/min/mg. The subunit molecular mass is 53 kDa, and the native molecular mass is 210 kDa, suggesting a tetrameric structure. Soluble high Km 5'-nucleotidase is most active with IMP and GMP and their deoxy derivatives. IMP is hydrolyzed 15 times faster than AMP. The enzyme has a virtually absolute requirement for magnesium ions and is regulated by them. Purine nucleoside 5'-triphosphates strongly activate the enzyme with the potency order dATP greater than ATP greater than GTP. 2,3-Diphosphoglycerate activates the enzyme as potently as ATP. Three millimolar ATP decreased the Km for IMP from 0.33 to 0.09 mM and increased the Vmax 12-fold. ATP activation was modified by the IMP concentration. At 20 microM IMP the ATP-dependent activation curve was sigmoidal, while at 2 mM IMP it was hyperbolic. The A0.5 values for ATP were 2.26 and 0.70 mM, and the relative maximal velocities were 32.9 and 126.0 nmol/min, respectively. Inorganic phosphate shifts the hyperbolic substrate velocity relationship for IMP to a sigmoidal one. With physiological concentrations of cofactors (3 mM ATP, 1-4 mM Pi, 150 mM KCl) at pH 7.4, the enzyme is 25-35 times more active toward 100 microM IMP than 100 microM AMP. These data show that: (a) soluble human placental high Km 5'-nucleotidase coexists in human placenta with the low Km enzyme; (b) under physiological conditions the enzyme favors the hydrolysis of IMP and is critically regulated by IMP, ATP, and Pi levels; and (c) kinetic properties of ATP and IMP are each modified by the other compound suggesting complex interaction of the associated binding sites.     

Human hypoxanthine-guanine phosphoribosyltransferase. Steady state kinetics of the forward and reverse reactions

A steady state kinetic study of the hypoxanthine-guanine phosphoribosyltransferase-catalyzed reaction in the forward and the reverse directions was carried out. The results obtained favor a sequential mechanism where the monomagnesium complexes of IMP and PPi bind to the enzyme in a rapid equilibrium random fashion while products must dissociate from the enzyme in ordered sequence, first the purine base and then the magnesium complex(es) of P-Rib-PP.     

Human brain hypoxanthine guanine phosphoribosyltransferase: structural and functional comparison with erythrocyte hypoxanthine guanine phosphoribosyltransferase

A rapid and simple method, based on GMP Sepharose affinity chromatography, was used for the purification of human brain hypoxanthine guanine phosphoribosyltransferase. A single protein band was detected by polyacrylamide gel electrophoresis of the native purified enzyme. A subunit molecular weight of 25,000 was estimated by SDS gel electrophoresis. The Km values for hypoxanthine and phosphoribosyl pyrophosphate were 50 and 111 microM, respectively. The Ki values for GMP and IMP with phosphoribosyl pyrophosphate were 21 and 37 microM, respectively. The purified enzyme from human brain did not differ significantly from the human erythrocyte one in amino acid composition. The brain and erythrocyte hypoxanthine guanine phosphoribosyltransferases showed complete immunochemical identity on Ouchterlony double diffusion.     

Purine base transport in normal and mutant erythrocytes.

The uptake of adenine and hypoxanthine in HGPRT-deficient and normal human erythrocytes was measured using a rapid filtering centrifugation technique. The transport of hypoxanthine as well as of adenine is impaired in the mutant cells. The transport of hypoxanthine into HGPRT-deficient erythrocytes differs from that into normal cells with respect to a higher accumulation capacity, to lower initial velocities and to the kinetic properties of the translocator. In addition, a higher accumulation capacity and lower initial velocities of adenine uptake could be demonstrated in mutant cells. A linkage of the purine translocator with purine phosphoribosyltransferases associated with the erythrocyte membrane is discussed.     

Regulation of 5-phosphoribosyl 1-pyrophosphate and of hypoxanthine uptake and release in human erythrocytes by oxypurine cycling.

Uptake and release of purines by red blood cells has been shown to be markedly sensitive to changes in pH, inorganic phosphate (Pi), and oxygen concentration (Berman, P., Black, D., Human, L., and Harley, E. (1988) J. Clin. Invest. 82, 980-986). The mechanism of this regulation has been further studied. We have shown that incubation of red cells in medium containing xanthine oxidase rapidly and completely depletes intracellular hypoxanthine and causes accumulation of 5-phosphoribosyl 1-pyrophosphate (PRPP) at physiological Pi concentrations. Hypoxanthine release from intracellular IMP is strictly dependent on PRPP depletion, induced by either alkalinizing the cells or by adding excess adenine. Xanthine oxidase abolishes this dependence. Oxygen depletion enhances adenine uptake and prevents hypoxanthine release. The results suggest that hypoxanthine release is governed by PRPP-dependent recycling of hypoxanthine to IMP. We propose that PRPP accumulation in red cells is regulated by a substrate cycle, comprising hypoxanthine, IMP, and inosine. Cycle flux is controlled by Pi inhibition and 2,3-bisphosphoglycerate activation of purine-5'-nucleotidase, which converts IMP to inosine. Oxypurine cycling may account for the sensitive control of purine uptake and release by changes in pH and oxygen tension that occur physiologically.     

Accelerated degradation of adenine nucleotide in erythrocytes of patients with chronic renal failure

Recently, we have shown that erythrocytes obtained from patients with chronic renal failure (CRF) exhibited an increased rate of ATP formation from adenine as a substrate. Thus, we concluded that this process was in part responsible for the increase of adenine nucleotide concentration in uremic erythrocytes. There cannot be excluded however, that a decreased rate of adenylate degradation is an additional mechanism responsible for the elevated ATP concentration. To test this hypothesis, in this paper we compared the rate of adenine nucleotide breakdown in the erythrocytes obtained from patients with CRF and from healthy subjects.Using HPLC technique, we evaluated: (1) hypoxanthine production by uremic RBC incubated in incubation medium: (a) pH 7.4 containing 1.2 mM phosphate (which mimics physiological conditions) and (b) pH 7.1 containing 2.4 mM phosphate (which mimics uremic conditions); (2) adenine nucleotide degradation (IMP, inosine, adenosine, hypoxanthine production) by uremic RBC incubated in the presence of iodoacetate (glycolysis inhibitor) and EHNA (adenosine deaminase inhibitor). The erythrocytes of healthy volunteers served as control.The obtained results indicate that adenine nucleotide catabolism measured as a hypoxanthine formation was much faster in erythrocytes of patients with CRF than in the cells of healthy subjects. This phenomenon was observed both in the erythrocytes incubated at pH 7.4 in the medium containing 1.2 mM inorganic phosphate and in the medium which mimics hyperphosphatemia (2.4 mM) and metabolic acidosis (pH 7.1). The experiments with EHNA indicated that adenine nucleotide degradation proceeded via AMP-IMP-Inosine-Hypoxanthine pathway in erythrocytes of both patients with CRF and healthy subjects. Iodoacetate caused a several fold stimulation of adenylate breakdown. Under these conditions: (a) the rate of AMP catabolites (IMP + inosine + adenosine + hypoxanthine) formation was substantially higher in the erythrocytes from patients with CRF; (b) in erythrocytes of healthy subjects degradation of AMP proceeded via IMP and via adenosine essentially at the same rate; (c) in erythrocytes of patients with CRF the rate of AMP degradation via IMP was about 2 fold greater than via adenosine.The results presented in this paper suggest that adenine nucleotide degradation is markedly accelerated in erythrocytes of patients with CRF.     

Distinct mechanisms of hypoxanthine and inosine transport in membrane vesicles isolated from Chinese hamster ovary and Balb 3T3 cells

Both enzyme-mediated group translocation and facilitated diffusion have been proposed as mechanisms by which mammalian cells take up purine bases and nucleosides. We have investigated the mechanisms for hypoxanthine and inosine transport by using membrane vesicles from Chinese hamster ovary cells (CHO), Balb/c 3T3 and SV3T3 cells prepared by identical procedures. Uptake mechanisms were characterized by analyzing intravesicular contents, determining which substrates could exchange with the transport products, assaying for hypoxanthine phosphoribosyltransferase activity, and measuring the stimulation of uptake of hypoxanthine by phosphoribosyl pyrophosphate ($\text{P}\text{Rib}\text{-PP}$).We found that the uptake of hypoxanthine in Balb 3T3 vesicles was stimulated 3–4-fold by $\text{P}\text{Rib}\text{-PP}$. The intravesicular product was predominantly IMP. The hypoxanthine phosphoribosyltransferase activity copurified with the vesicle preparation. These results suggest the possible involvement of this enzyme in hypoxanthine uptake in 3T3 vesicles. In contrast to the 3T3 vesicles, CHO vesicles prepared under identical procedures did not retain hypoxanthine phosphoribosyltransferase activity and did not demonstrate $\text{P}\text{Rib}\text{-PP}$-stimulated hypoxanthine uptake. The intravesicular product of hypoxanthine uptake in CHO vesicles was hypoxanthine. These results and data from our kinetic and exchange studies indicated that CHO vesicles transport hypoxanthine via facilitated diffusion. An analogous situation was observed for inosine uptake; CHO vesicles accumulated inosine via a facilitated diffusion mechanism, while in the same experiments SV3T3 vesicles exhibited a purine nucleoside phosphorylase-dependent translocation of the ribose moiety of inosine.     

Phosphorothioate analogues of 5-phosphoribosyl 1-diphosphate: synthesis, purification, and partial characterization

The 1-phosphorothioate analogues of 5-phosphoribosyl 1-diphosphate (P-Rib-PP) have been prepared enzymatically, in reactions catalyzed by P-Rib-PP synthetase from Salmonella typhimurium. 5-Phosphoribosyl 1-O-(2-thiodiphosphate) (P-Rib-PP beta S) was synthesized from ribose 5-phosphate (Rib-5-P) and the Mg2+ complex of adenosine 5'-O-(3-thiotriphosphate). The SP and RP diastereomers of 5-phosphoribosyl 1-O-(1-thiodiphosphate) (P-Rib-PP alpha S) were synthesized from Rib-5-P and the Mg2+ complex of adenosine 5'-O-(2-thiotriphosphate) (ATP beta S) (SP diastereomer, delta-configuration) and the Cd2+ complex of ATP beta S (RP diastereomer, delta-configuration), respectively. The strategy for the synthesis and stereochemical assignment of the P-Rib-PP alpha S diastereomers was based on the specificity of P-Rib-PP synthetase for the (delta)-beta, gamma-bidentate metal-nucleotide substrate and the stereochemical course of the synthetase reaction, leading to inversion of configuration at the P beta atom of the nucleotide [Li, T. M., Mildvan, A. S., & Switzer, R. L. (1978) J. Biol. Chem. 253, 3918-3923], and the known configurations of the Mg2+ and Cd2+ beta, gamma-bidentate complexes of the ATP beta S diastereomers [Jaffe, E. K., & Cohn, M. (1979) J. Biol. Chem. 254, 10839-10845]. The P-Rib-PP analogues were purified by gradient elution from DEAE-Sephadex and characterized by chemical analysis and 31P nuclear magnetic resonance [Smithers, G. W., & O'Sullivan, W. J. (1984) Biochemistry (following paper in this issue)]. A preliminary account of their interaction with human brain hypoxanthine phosphoribosyltransferase and yeast orotate phosphoribosyltransferase (OPRTase) is described.(ABSTRACT TRUNCATED AT 250 WORDS)     

A very fast ion-pair reversed-phase HPLC method for the separation of the most significant nucleotides and their degradation products in human red blood cells

A simple and fast ion pair reversed-phase high-performance liquid chromatographic method has been developed for the simultaneous determination of ATP, ADP, AMP, GTP, GDP, IMP, NADP+, NADPH+, NAD+, NADH, ADP-ribose, inosine, adenosine, hypoxanthine, and xanthine. This method allows us to have a complete picture of the most important nucleotides present in fresh human erythrocytes. Furthermore it is particularly useful in the study of the erythrocyte adenine nucleotide catabolism allowing the detection of degradation products such as IMP, inosine, adenosine, hypoxanthine, and xanthine. The separation of the compounds under investigation is achieved in less than 15 min using a reversed-phase 3-micron Supelcosil LC-18 column and adding tetrabutylammonium, as ion-pair agent, to the buffers. The short time of analysis, the high reproducibility of the system, and the accurate evaluation of the compounds of interest make this method particularly suitable for routine analysis. Finally it is possible to use this assay as an alternative method of measuring activities of enzymes which catalyze reactions involving some of these compounds, as in the case of Na+-K+ ATPase, AMP deaminase, and adenosine deaminase.     

A straightforward radiometric technique for measuring IMP dehydrogenase

[2-3H]Inosinic acid ([2-3H]IMP) has been biosynthesized in good yield from [2-3H]hypoxanthine and PRPP via the action of a partially purified preparation of hypoxanthine/guanine phosphoribosyl transferase from mouse brain. The product was purified in one step by ascending paper chromatography, and used to assess the activity of IMP dehydrogenase. To conduct the assay, tritiated substrate is admixed with enzyme in a final volume of 10 microliters; NAD is present to serve as cofactor for the reaction, and allopurinol to inhibit the oxidation of any hypoxanthine generated as a consequence of side reactions. After an appropriate period of incubation, the 3H2O arising from the oxidation of tritiated IMP via [3H]NAD is isolated by quantitative microdistillation. Performed as described, the assay is facile, sensitive, and accurate, with the capability of detecting the dehydrogenation of as little as 1 pmol of [3H]IMP. Using it, measurements have been made of IMP dehydrogenase in a comprehensive array of mouse organs. Of these, pancreas contained the enzyme at the highest specific activity.