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At least 19 recordsLinked to original sources
PubMed1990

Effect of dipyridamole on inosine triphosphate pyrophosphohydrolase activity and inosine triphosphate content in fresh human erythrocytes incubated with adenosine.

The activity of inosine triphosphate pyrophosphohydrolase (ITPH) in human erythrocytes was found to be 1.50 +/- 0.39 mumol of inosine triphosphate (ITP) hydrolysed x min-1 per g Hb, and no measurable amount of ITP was detected. When dipyridamole was added to the medium composed of adenosine, pyruvate and inorganic phosphate, ITPH activity was 1.18 +/- 0.41, and at the same time ITP accumulation was 0.61 +/- 0.31 mumol/g Hb. The negative correlation between ITPH activity and accumulation of ITP was r = -0.87 at P less than 0.001.

Adenosine↗
PubMed1997

Functionally nonequivalent interactions of guanosine 5'-triphosphate, inosine 5'-triphosphate, and xanthosine 5'-triphosphate with the retinal G-protein, transducin, and with Gi-proteins in HL-60 leukemia cell membranes.

G-proteins mediate signal transfer from receptors to effector systems. In their guanosine 5'-triphosphate (GTP)-bound form, G-protein alpha-subunits activate effector systems. Termination of G-protein activation is achieved by the high-affinity GTPase [E.C. 3.6.1.-] of their alpha-subunits. Like GTP, inosine 5'-triphosphate (ITP) and xanthosine 5'-triphosphate (XTP) can support effector system activation. We studied the interactions of GTP, ITP, and XTP with the retinal G-protein, transducin (TD), and with G-proteins in HL-60 leukemia cell membranes. TD hydrolyzed nucleoside 5'-triphosphates (NTPs) in the order of efficacy GTP > ITP > XTP. NTPs eluted TD from rod outer segment disk membranes in the same order of efficacy. ITP and XTP competitively inhibited TD-catalyzed GTP hydrolysis. In HL-60 membranes, the chemoattractants N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) and leukotriene B4 (LTB4) effectively activated GTP and ITP hydrolysis by Gi-proteins. fMLP and LTB4 were at least 10-fold more potent activators of ITPase than of GTPase. Complement C5a effectively activated the GTPase of Gi-proteins but was only a weak stimulator of ITPase. The potency of C5a to activate GTP and ITP hydrolysis was similar. The fMLP-stimulated GTPase had a lower Km value than the fMLP-stimulated ITPase, whereas the opposite was true for the Vmax values. fMLP, C5a, and LTB4 did not stimulate XTP hydrolysis. Collectively, our data show that GTP, ITP, and XTP bind to G-proteins with different affinities, that G-proteins hydrolyze NTPs with different efficacies, and that chemoattractants stimulate GTP and ITP hydrolysis by Gi-proteins in a receptor-specific manner. On the basis of our results and the data in the literature, we put forward the hypothesis that GTP, ITP, and XTP act as differential signal amplifiers and signal sorters at the G-protein level.

Animals↗
PubMed1977

Temperature dependence of the decay of the UV absorption difference spectrum of heavy meromyosin induced by adenosine triphosphate and inosine triphosphate.

The UV absorption difference spectrum of heavy meromyosin induced by ATP was measured at various temperatures. At higher temperatures, the difference spectrum formed rapidly after adding ATP and continued steadily during the steady state which we have called the ATP-form of difference spectrum. At lower temperatures, the ATP-form of difference spectrum decayed into the other form before the steady state was attained. This was identical to the difference spectrum obtained by adding ADP and has been called the ADP-form of difference spectrum. At intermediate temperatures, biphasic decay was observed. The results indicate that the dominant intermediate at the steady state is altered from the one showing the ATP-form of difference spectrum at higher temperatures to that showing the ADP-form at lower temperatures. The population of the two intermediates depends on the temperature between the two extremes. This temperature-induced transition was observed in the presence of any divalent cation such as Mg2+, Mn2+, or Ca2+. A similar transition was observed with the difference spectrum induced by ITP in the presence of MgCl2. The pH dependence of the single early decay of the ATP-induced difference spectrum was measured in the presence of MnCl2 at 1 degree. The apparent rate constant of the decay showed a biphasic pH dependence, having the same shape as the pH activity curve of ATPase [EC 3.6.1.3] observed at higher temperatures. The rate determining step for the steady state ATPase at higher temperatures is thought to be the step of changing from the intermediate complex showing the ATP-form of difference spectrum to that showing the ADP-form. This is inconsistent with our previous mechanism (Yazawa, M. et al. (1973) J. Biochem. 74, 1107-1117). The rate determining step at lower temperatures was assigned as a step of ADP dissociation.

Adenosine Diphosphate↗
PubMed1979

Purification and properties of human erythrocyte inosine triphosphate pyrophosphohydrolase.

Inosine triphosphate pyrophosphohydrolase from human erythrocytes was purified and characterized. The enzyme is highly specific for ITP and shows optimal activity in glycine buffer pH 9.6 and 50 mM MgCl2. The Km of the enzyme is 1.3 X 10(-4), the Vmax = 1.2 X 10(-9) and the Keq = 3.8 X 10(4). Human erythrocyte ITP pyrophosphohydrolase does not require SH compounds for activation. The enzyme is inhibited by Cd++, Co++, and Ca++ ions and by p-hydroxymercuribenzoate.

Cations, Divalent↗
PubMed1979

Nonparallel isometric tension response of rabbit soleus skinned muscle fibers to magnesium adenosine triphosphate and magnesium inosine triphosphate.

The isometric tension response of single "skinned' rabbit soleus muscle fibers to MgATP and McITP in the absence of calcium was studied. [MgATP] or [MgITP] was varied in solutions of ionic strength 0.30 and temperature 20 degrees C. Steady-state tension that developed in MgATP or MgITP solutions was a biphasic bell-shaped function of log [MgATP] or log [MgITP] which increased from zero to maximum tension and then declined again to zero. Analysis of the data showed that, under comparable ionic conditions, percent tension vs. log [MgATP] and percent tension vs. log [MgITP] curves are not parallel. Instead, the percent tension vs. log [MgITP] curve is much broader. Additionally, under comparable ionic conditions maximum tension in MgITP solutions was higher than in MgATP solutions. In addition, in MgATP solutions, pH, [K+], and excess ATP were varied. Raising pH from 7 to 8, [K+] from 46 mM to 200 mM, or decreasing excess ATP from 2 to 0.5 mM all increased maximum tension. None of these factors, however, influenced the shape or position of the percent tension vs. log [MgATP] curve.

Adenosine Triphosphate↗
PubMed1976

Relationships between nucleoside triphosphate pyrophosphohydrolase activity and inosine triphosphate accumulation in human erythrocytes.

The relationship between nucleoside triphosphate pyrophosphohydrolast (NTPH) (EC 3.6.1.19) activity in erythrocyte lysates and accumulation of radioactive inosine triphosphate (ITP) in human erythrocytes incubated in vitro with [14C]hypoxanthine, was studied in 93 humans. When ITP accumulation, expressed as percentage of total radioactive nucleotides, was plotted against NTPH specific activity, an inverse relationship was found to exist. A continous spectrum of NTPH specific activities and ITP accumulation values exists in the human population and the relationship between these two parameters follows the relationship of substrate concentration to enzyme activity predicted by Michaelis-Menten enzyme kinetics. One interpretation of these data is that the ITP concentration in human red blood cells is controlled by the degradation of ITP to IMP and pyrophosphate catalyzed by NTPH.

Erythrocytes↗
PubMed2002

Genetic basis of inosine triphosphate pyrophosphohydrolase deficiency.

Inosine triphosphate pyrophosphohydrolase (ITPase) deficiency is a common inherited condition characterized by the abnormal accumulation of inosine triphosphate (ITP) in erythrocytes. The genetic basis and pathological consequences of ITPase deficiency are unknown. We have characterized the genomic structure of the ITPA gene, showing that it has eight exons. Five single nucleotide polymorphisms were identified, three silent (138G-->A, 561G-->A, 708G-->A) and two associated with ITPase deficiency (94C-->A, IVS2+21A-->C). Homozygotes for the 94C-->A missense mutation (Pro32 to Thr) had zero erythrocyte ITPase activity, whereas 94C-->A heterozygotes averaged 22.5% of the control mean, a level of activity consistent with impaired subunit association of a dimeric enzyme. ITPase activity of IVS2+21A-->C homozygotes averaged 60% of the control mean. In order to explore further the relationship between mutations and enzyme activity, we examined the association between genotype and ITPase activity in 100 healthy controls. Ten subjects were heterozygous for 94C-->A (allele frequency: 0.06), 24 were heterozygotes for IVS2+21A-->C (allele frequency: 0.13) and two were compound heterozygous for these mutations. The activities of IVS2+21A-->C heterozygotes and 94C-->A/IVS2+21A-->C compound heterozygotes were 60% and 10%, respectively, of the normal control mean, suggesting that the intron mutation affects enzyme activity. In all cases when ITPase activity was below the normal range, one or both mutations were found. The ITPA genotype did not correspond to any identifiable red cell phenotype. A possible relationship between ITPase deficiency and increased drug toxicity of purine analogue drugs is proposed.

Adult↗
PubMed1998

Identification of stable RNA hairpins causing band compression in transcriptional sequencing and their elimination by use of inosine triphosphate.

To identify stable RNA secondary structure causing band compression, 30 lambda DNA clones and four cDNA clones (about 10 kb in total length) were sequenced using Transcriptional Sequencing, which is based on the phage RNA polymerase chain termination reaction with fluorescent 3' deoxynucleoside triphosphate, using the canonical set of rNTPs for the substrate. Electrophoresis was performed on acrylamide gel containing 7 M urea at 50 degrees C using ABI 377 DNA sequencer. A total of 159 band compressions were identified, and most compression sites seem to be due to hairpin structures. We also found that the presence of rITP in place of rGTP in the sequencing reaction can entirely eliminate all band compressions. The use of rITP gave a better peak uniformity and resolution in the sequencing gel in the case of lambda DNA than with c7rGTP, leading to improved accuracy in the sequence determination. Substitution of the base analog rITP for rGTP should be useful for accurate sequencing determination.

Bacteriophage lambda↗
PubMed2005

Genetic basis of inosine triphosphate pyrophosphohydrolase deficiency in the Japanese population.

Inosine triphosphate pyrophosphohydrolase (ITPase) is an enzyme that catalyzes the conversion of inosine triphosphate (ITP) to inosine monophosphate and pyrophosphate. In Caucasian populations it is reported that the frequency of cases showing decreased ITPase activity is 5%. The structure of ITPA gene along with five single nucleotide polymorphisms has been reported in Caucasians. We examined ITPase activity and frequency of two polymorphisms (94C>A and IVS2+21A>C) in 100 Japanese individuals. Among these individuals, we observed that three cases with zero activity were homozygote for 94C>A, and were accompanied by abnormal accumulation of ITP in erythrocytes. The cases included in the low ITPase activity group were heterozygote for 94C>A polymorphism. The activity of the heterozygote cases was approximately 27% of the mean value of the wild type. The allele frequency of the 94C>A polymorphism was 0.155, which was 2.6 times higher than that of the Caucasians (0.06). The IVS2+21A>C was not detected in Japanese cases, although it occurred with a frequency of 0.130 in Caucasians. Furthermore, we identified a novel mutation IVS2+68T>G in intron 2 in the case with the lowest enzyme activity in the 94C>A wild type. Since the frequency of ITPA 94C>A polymorphism is higher in the Japanese population than that in Caucasians, it is more important to examine ITPA 94C>A polymorphism in the Japanese population to prevent thiopurine drug toxicity. Pretherapeutic screening of individuals for ITPA polymorphisms should be considered for safer and more tolerable treatment with thiopurine drugs.

Adult↗
PubMed1975

Individual variation in inosine triphosphate accumulation in human erythrocytes.

Erythrocytes from 5% of a normal population accumulated relatively high amounts of radioactive inosine triphosphate (i.e., greater than 70 nmoles/10(10) cells in 2 hr) when they were incubated with [14C]hypoxanthine. The incidence of this characteristic in a mentally retarded population was 16%. Inosine triphosphate was synthesized from [14C]hypoxanthine, but not from [14C]adenine or [14C]guanine. The metabolism of [14C]adenine and [14C]guanine was the same in erythrocytes that accumulated "normal" and "high" amounts of inosine triphosphate. Inosine triphosphate did not accumulate in leukocytes.

Adenine↗
PubMed1981

Activity of inosine triphosphate pyrophosphohydrolase in fresh and stored human erythrocytes.

The accumulation of inosine triphosphate (ITP) in human erythrocytes incubated with inosine depends on the activity of inosine triphosphate pyrophosphohydrolase (ITPH). High activity of this enzyme is accompanied by a low concentration of ITP and conversely. We showed that ITPH activity decreases with the prolongation of blood preservation time. As a consequence there is a lower accumulation of ITP in fresh erythrocytes incubated in a medium containing high concentrations of inosine, pyruvate and phosphate (IPP) than in red blood cells preserved at 4 degrees C. Synthesis of ITP in erythrocytes incubated in IPP medium being so intensive, it seems possible that during incubation an intermediate accumulates which decreases ITPH activity.

Blood Preservation↗
PubMed1977

Factors affecting inosinate synthesis and inosine triphosphate accumulation in human erythrocytes.

Measurements of rates of inosinate synthesis from radioactive hypoxanthine by human erythrocytes show a large degree of individual variation. Rates of inosinate synthesis also vary with the pH and phosphate concentration of the incubation medium. This may be due to changes in the rate of phosphoribosyl pyrophosphate synthesis, and the stimulatory effect of phosphate on this process seems to be more important than the inhibitory effect of 2,3-diphodphoglycerate. The rate of inosinate synthesis, and especially the extent of accumulation of inosine triphosphate, increase disproportionately with time of incubation up to at least 24 h. Storage of erythrocytes also tends to increase inosinate synthesis and inosine triphosphate accumulation.

Anaerobiosis↗
PubMed2006

Structure of the orthorhombic form of human inosine triphosphate pyrophosphatase.

The structure of human inosine triphosphate pyrophosphohydrolase (ITPA) has been determined using diffraction data to 1.6 A resolution. ITPA contributes to the accurate replication of DNA by cleansing cellular dNTP pools of mutagenic nucleotide purine analogs such as dITP or dXTP. A similar high-resolution unpublished structure has been deposited in the Protein Data Bank from a monoclinic and pseudo-merohedrally twinned crystal. Here, cocrystallization of ITPA with a molar ratio of XTP appears to have improved the crystals by eliminating twinning and resulted in an orthorhombic space group. However, there was no evidence for bound XTP in the structure. Comparison with substrate-bound NTPase from a thermophilic organism predicts the movement of residues within helix alpha1, the loop before alpha6 and helix alpha7 to cap off the active site when substrate is bound.

Crystallography, X-Ray↗