Inosine 5'-diphosphate, a molecular decoy rescues Nucleoside diphosphate kinase from c-MYC G-Quadruplex unfolding.

BACKGROUND: The transcription-inhibitory G-Quadruplex(Pu27-GQ) at c-MYC promoter is challenging to target due to structural heterogeneity. Nucleoside diphosphate kinase (NM23-H2) specifically binds and unfolds Pu27-GQ to increase c-MYC transcription. Here, we used Inosine 5'-diphosphate (IDP) to disrupt NM23-H2-Pu27-GQ interactions and arrest c-MYC transcription without compromising NM23-H2-mediated kinase properties. METHODS: Site-directed mutagenesis,31P-NMR and STD-NMR studies delineate the epitope of NM23-H2-IDP complex and characterize specific amino acids in NM23-H2 involved in Pu27-GQ and IDP interactions. Immunoprecipitations and phosphohistidine-immunoblots reveal how IDP blocks NM23-H2-Pu27 association to downregulate c-MYC transcription in MDAMB-231 cells exempting NM23-H2-mediated kinase properties. RESULTS: NMR studies show that IDP binds to the Guanosine diphosphate-binding pocket of NM23-H2 (KD = 5.0 ± 0.276 µM). Arg88-driven hydrogen bonds to the terminal phosphate of IDP restricts P-O-P bond-rotation increasing its pKa (∆pKa = 0.85 ± 0.0025).9-inosinyl moiety of IDP is stacked over Phe60 phenyl ring driving trans-conformation of inosine and axial geometry of pyrophosphates. Chromatin immunoprecipitations revealed that these interactions rescue NM23-H2-driven Pu27-GQ unfolding, which triggers Nucleolin recruitment and lowers Sp1 occupancy at c-MYC promoter stabilizing Pu27-GQ. This silences c-MYC transcription that reduces c-MYC-Sp1 association amplifying Sp1 recruitment across P21 promoter stimulating P21 transcription and G2/M arrest. CONCLUSIONS: IDP synergizes the effects of Pu27-GQ-interacting compounds to abrogate c-MYC transcription and induce apoptosis in MDAMB-231 cells by disrupting NM23-H2-Pu27-GQ interactions without affecting NM23-H2-mediated kinase properties. GENERAL SIGNIFICANCE: Our study provides a pragmatic approach for developing NM23-H2-targeting regulators to rescue NM23-H2 binding at structurally ambiguous Pu27-GQ that synergizes the anti-tumorigenic effects of GQ-based therapeutics with minimized off-target effects.

Integrating All-Atom and Coarse-Grained Simulations-Toward Understanding of IDPs at Surfaces.

We present a scheme for transferring conformational degrees of freedom from all-atom (AA) simulations of an intrinsically disordered protein (IDP) to coarse-grained (CG) Monte Carlo (MC) simulations using conformational swap moves. AA simulations of a single histatin 5 peptide in water were used to obtain a structural ensemble, which is reweighted in a CGMC simulation in the presence of a negatively charged surface. For efficient sampling, the AA trajectory was condensed using two approaches: RMSD clustering (based on the root-mean-square difference in atom positions) and a "naïve" truncation, where only every 100th frame of the trajectory was included in the library. The results show that even libraries with few structures well reproduce the radius of gyration and interaction free energy as functions of the distance from the surface. We further observe that the surface slightly promotes the secondary structure of histatin 5 and more so if using explicit surface charges rather than smeared charges.

Different substrate specificities of the two ADPR binding sites in TRPM2 channels of Nematostella vectensis and the role of IDPR.

NvTRPM2 (Nematostella vectensis Transient Receptor Potential Melastatin 2), the species variant of the human apoptosis-related cation channel hTRPM2, is gated by ADP-ribose (ADPR) independently of the C-terminal NUDT9H domain that mediates ADPR-directed gating in hTRPM2. The decisive binding site in NvTRPM2 is likely to be identical with the N-terminal ADPR binding pocket in zebra fish DrTRPM2. Our aim was a characterization of this binding site in NvTRPM2 with respect to its substrate specificity, in comparison to the classical ADPR interaction site within NUDT9H that is highly homologous in hTRPM2 and NvTRPM2, although only in NvTRPM2, catalytic (ADPRase) activity is conserved. With various ADPR analogues, key differences of the two sites were identified. Particularly, two reported antagonists on hTRPM2 were agonists on NvTRPM2. Moreover, IDP-ribose (IDPR) induced currents both in hTRPM2 and NvTRPM2 but not in NvTRPM2 mutants in which NUDT9H was absent. Thus, IDPR acts on NUDT9H rather than N-terminally, revealing a regulatory function of NUDT9H in NvTRPM2 opposed to that in hTRPM2. We propose that IDPR competitively inhibits the ADPRase function of NUDT9H and evokes ADPR accumulation. The findings provide important insights into the structure-function relationship of NvTRPM2 and will allow further characterization of the novel ADPR interaction site.

Second messenger analogues highlight unexpected substrate sensitivity of CD38: total synthesis of the hybrid "L-cyclic inosine 5'-diphosphate ribose".

The multifunctional, transmembrane glycoprotein human CD38 catalyses the synthesis of three key Ca2+-mobilising messengers, including cyclic adenosine 5'-diphosphate ribose (cADPR), and CD38 knockout studies have revealed the relevance of the related signalling pathways to disease. To generate inhibitors of CD38 by total synthesis, analogues based on the cyclic inosine 5'-diphosphate ribose (cIDPR) template were synthesised. In the first example of a sugar hybrid cIDPR analogue, "L-cIDPR", the natural "northern" N1-linked D-ribose of cADPR was replaced by L-ribose. L-cIDPR is surprisingly still hydrolysed by CD38, whereas 8-Br-L-cIDPR is not cleaved, even at high enzyme concentrations. Thus, the inhibitory activity of L-cIDPR analogues appears to depend upon substitution of the base at C-8; 8-Br-L-cIDPR and 8-NH2-L-cIDPR inhibit CD38-mediated cADPR hydrolysis (IC50 7 µM and 21 µM respectively) with 8-Br-L-cIDPR over 20-fold more potent than 8-Br-cIDPR. In contrast, L-cIDPR displays a comparative 75-fold reduction in activity, but is only ca 2-fold less potent than cIDPR itself. Molecular modelling was used to explore the interaction of the CD38 catalytic residue Glu-226 with the "northern" ribose. We propose that Glu226 still acts as the catalytic residue even for an L-sugar substrate. 8-Br-L-cIDPR potentially binds non-productively in an upside-down fashion. Results highlight the key role of the "northern" ribose in the interaction of cADPR with CD38.

Phosphoenolpyruvate metabolism in Teladorsagia circumcincta: a critical junction between aerobic and anaerobic metabolism.

Nematodes which have adapted to an anaerobic lifestyle in their adult stages oxidise phosphoenolpyruvate (PEP) to oxaloacetate rather than pyruvate as the final product of glycolysis. This adaptation involves selective expression of the enzyme phosphoenolpyruvate carboxykinase (PEPCK), instead of pyruvate kinase (PK). However, such adaptation is not absolute in aerobic nematode species. We have examined the activity and kinetics of PEPCK and PK in larvae (L(3)) and adults of Teladorsagia circumcincta, a parasite known to exhibit oxygen uptake. Results revealed that PK and PEPCK activity existed in both L(3)s and adults. The enzymes had differing affinity for nucleotide diphosphates: while both can utilise GDP, only PK utilised ADP and only PEPCK utilised IDP. In both life cycle stages, enzymes showed similar affinity for PEP. PK activity was predominant in both stages, although activity of this enzyme was lower in adults. When combined, both the activity levels and the enzyme kinetics showed that pyruvate production is probably favoured in both L(3) and adult stages of T. circumcincta and suggest that metabolism of PEP to oxaloacetate is a minor metabolic pathway in this species.

NUDT16 and ITPA play a dual protective role in maintaining chromosome stability and cell growth by eliminating dIDP/IDP and dITP/ITP from nucleotide pools in mammals.

Mammalian inosine triphosphatase encoded by ITPA gene hydrolyzes ITP and dITP to monophosphates, avoiding their deleterious effects. Itpa(-) mice exhibited perinatal lethality, and significantly higher levels of inosine in cellular RNA and deoxyinosine in nuclear DNA were detected in Itpa(-) embryos than in wild-type embryos. Therefore, we examined the effects of ITPA deficiency on mouse embryonic fibroblasts (MEFs). Itpa(-) primary MEFs lacking ITP-hydrolyzing activity exhibited a prolonged doubling time, increased chromosome abnormalities and accumulation of single-strand breaks in nuclear DNA, compared with primary MEFs prepared from wild-type embryos. However, immortalized Itpa(-) MEFs had neither of these phenotypes and had a significantly higher ITP/IDP-hydrolyzing activity than Itpa(-) embryos or primary MEFs. Mammalian NUDT16 proteins exhibit strong dIDP/IDP-hydrolyzing activity and similarly low levels of Nudt16 mRNA and protein were detected in primary MEFs derived from both wild-type and Itpa(-) embryos. However, immortalized Itpa(-) MEFs expressed significantly higher levels of Nudt16 than the wild type. Moreover, introduction of silencing RNAs against Nudt16 into immortalized Itpa(-) MEFs reproduced ITPA-deficient phenotypes. We thus conclude that NUDT16 and ITPA play a dual protective role for eliminating dIDP/IDP and dITP/ITP from nucleotide pools in mammals.

Unusual features of a recombinant apple alpha-farnesene synthase.

A recombinant alpha-farnesene synthase from apple (Malus x domestica), expressed in Escherichia coli, showed features not previously reported. Activity was enhanced 5-fold by K(+) and all four isomers of alpha-farnesene, as well as beta-farnesene, were produced from an isomeric mixture of farnesyl diphosphate (FDP). Monoterpenes, linalool, (Z)- and (E)-beta-ocimene and beta-myrcene, were synthesised from geranyl diphosphate (GDP), but at 18% of the optimised rate for alpha-farnesene synthesis from FDP. Addition of K(+) reduced monoterpene synthase activity. The enzyme also produced alpha-farnesene by a reaction involving coupling of GDP and isoprenyl diphosphate but at <1% of the rate with FDP. Mutagenesis of active site aspartate residues removed sesquiterpene, monoterpene and prenyltransferase activities suggesting catalysis through the same active site. Phylogenetic analysis clusters this enzyme with isoprene synthases rather than with other sesquiterpene synthases, suggesting that it has evolved differently from other plant sesquiterpene synthases. This is the first demonstration of a sesquiterpene synthase possessing prenyltransferase activity.

[Nucleoside-5'-triphosphate hydrolysis in the liver and kidney of rats with chronic alloxan diabetes].

Activity and some properties of a soluble enzyme hydrolyzing nucleoside-5'-triphosphates were studied in the liver and kidney of normal and diabetic rats. The enzyme activity was shown to be reduced by 34% (p < 0.01) in the liver extracts of diabetic animals, while no difference was observed in the kidney. When ITP was used as substrate, the apparent Michaelis constant of the enzyme was significantly lower in the liver of controls as compared to experimental rats (32.3 +/- 1.3 microM and 54.3 +/- 1.0 microM, respectively, p < 0.01). The KM values of the enzyme in the kidney were not distinguishable in both groups. NTPase exhibits maximal activity at pH 7.0 and has a broad substrate specificity with respect to different nucleoside-5'-tri- and diphosphates. Molecular mass of the enzyme was estimated by gel filtration to be 63.7 +/- 0.9 kD.

Analysis of nucleotide binding by a vacuolar proton-translocating adenosine triphosphatase.

The vacuolar-type proton-translocatine adenosine triphosphatase from bovine adrenal secretory granules (chromaffin granules) was purified and reconstituted into proteoliposomes. The binding of nucleotides to the enzyme was studied by quantifying their effects on the rate of inactivation by N-ethylmaleimide (MalNEt) of ATP-dependent proton translocation, and by direct measurement of the binding of [3H]MgADP. The results of these experiments are consistent with a model of the enzyme that had been developed as a result of kinetic experiments, the features of which are that the enzyme exists in two states, each containing three nucleotide-binding sites on catalytic subunits, and that nucleoside diphosphates regulate the enzyme by binding with high affinity to a single site in the inactive T state of the enzyme. Under the conditions of the experiments, MalNEt inactivated the ATPase in a pseudo-first order reaction. Rate constants of inactivation were reduced in the presence of MgADP, MgIDP and free ADP; the kinetics of protection suggested that the two conformational states of the enzyme were inactivated at different rates and also confirmed the existence of two different types of binding site for MgADP. Low nucleotide concentrations afforded partial protection from MalNEt; this was ascribed to binding of nucleotide to the regulatory site causing a shift in the conformational equilibrium towards the T state, which was more slowly inactivated than the unliganded R state of the enzyme. At higher nucleotide concentrations, binding at the catalytic site afforded complete protection from MalNEt. Protection by MgADP[S] and magnesium 2'- and 3'-O-[4-benzoylbenzoyl]adenosine 5'-triphosphate showed simpler kinetics but was also consistent with previously reported kinetic results. Analysis of subunit labelling with [3H]MalNEt showed that the three 72-kDa (catalytic) subunits were alkylated by MalNEt with similar rate constants, consistent with a symmetrical arrangement of the catalytic subunits, in contrast to the situation in F-type ATPases. Analysis of the binding of [3H]MgADP also confirmed the results of kinetic experiments. MgADP was shown to bind to the enzyme with an apparent dissociation constant of about 66 nM; assuming that the nucleotide binds only to the T-state, the true dissociation constant is < 1 nM. Using Blue Native polyacrylamide gel electrophoresis to separate the holo-ATPase from the membrane sector, the stoichiometry of binding was calculated to be 0.6 mol/mol enzyme, confirming the existence of a single regulatory site for MgADP. However, binding of MgADP to the enzyme was much slower than could be accounted for by the measured dissociation constants, suggesting that it is rate limited by a step such as a protein conformational change. Treatment designed to remove endogenous nucleotide had no effect on the rate or extent of binding of MgADP.

Hydrogen bond interactions of G proteins with the guanine ring moiety of guanine nucleotides.

We have utilized Raman difference spectroscopy to investigate hydrogen bonding interactions of the guanine moiety in guanine nucleotides with the binding site of two G proteins, EF-Tu (elongation factor Tu from Escherichia coli) and the c-Harvey ras protein, p21 (the gene product of the human c-H-ras proto-oncogene). Raman spectra of proteins complexed with GDP (guanosine 5' diphosphate), IDP (inosine 5' diphosphate), 6-thio-GDP, and 6-18O-GDP were measured, and the various difference spectra were determined. These were compared to the difference spectra obtained in solution, revealing vibrational features of the nucleotide that are altered upon binding. Specifically, we observed significant frequency shifts in the vibrational modes associated with the 6-keto and 2-amino positions of the guanine group of GDP and IDP that result from hydrogen bonding interactions between these groups and the two proteins. These shifts are interpreted as being proportional to the local energy of interaction (delta H) between the two groups and protein residues at the nucleotide binding site. Consistent with the tight binding between the nucleotides and the two proteins, the shifts indicate that the enthalpic interactions are stronger between these two polar groups and protein than with water. In general, the spectral shifts provide a rationale for the stronger binding of GDP and IDP with p21 compared to EF-Tu. Despite the structural similarity of the binding sites of EF-Tu and p21, the strengths of the observed hydrogen bonds at the 6-keto and 2-amino positions vary substantially, by up to a factor of 2.(ABSTRACT TRUNCATED AT 250 WORDS)

Glutamine 170 to tyrosine substitution in yeast mitochondrial F1 beta-subunit increases catalytic site interaction with GDP and IDP and produces negative cooperativity of GTP and ITP hydrolysis.

Glutamine 170 to tyrosine mutation in the beta-subunit from Schizosaccharomyces pombe mitochondrial F1 was found to increase both affinity for ADP, apparent negative cooperativity of ATPase activity, and sensitivity to azide inhibition (Falson, P., Di Pietro, A., Jault, J.-M., Gautheron, D.C., and Boutry, M. (1989) Biochim. Biophys. Acta 975, 119-126). The mutation is shown here to increase the affinity for GDP, IDP, and guanosine 5'-(beta,gamma-imidotriphosphate), which are competitive inhibitors of GTPase and ITPase activities. Various fluorescence approaches also reveal an increased affinity of the catalytic site in mutant as compared with wild-type enzyme for GDP, IDP, and 2'(3')-N-methylanthraniloyl GDP. The mutation alters the maximal rates and pH dependence of GTPase and ITPase activities, whereas wild-type F1 exhibits single optima at pH 7.5-8.0. The pH activity profiles of the mutant enzyme for these substrates are biphasic, with optima at pH 8.5-9.0 and below 6.5. The mutation increases the sensitivity of GTPase and ITPase activities to azide inhibition, which increases with decreasing pH. At pH 6.0-7.0, an apparent negative cooperativity is observed when mutant F1 hydrolyzes GTP or ITP, whereas the wild-type enzyme shows Michaelian kinetics. Addition of bicarbonate at pH 7.0 substantially stimulates GTP or ITP hydrolysis and abolishes the apparent negative cooperativity by the mutant enzyme; on the contrary, the anion produces a slight inhibition of these activities catalyzed by wild-type F1. The overall results suggest that apparent negative cooperativity can be observed with GTP or ITP hydrolysis provided that the release of the respective diphosphate is a rate-limiting step.

Effects of inositol hexaphosphate on growth and differentiation in K-562 erythroleukemia cell line.

Inositol hexaphosphate (InsP6) has recently been shown to inhibit experimental cancers in vivo. Since the lower phosphorylated forms of InsP6 are important in cell growth in a wide variety of mammalian cells, we tested the efficacy of InsP6 in growth reduction of K-562 human erythroleukemia cells in vitro. We report that InsP6 decreases the K-562 cell population by 19-36% (P less than 0.001) concomitant to an increased differentiation as evidenced by ultrastructural morphology and increased hemoglobin synthesis. Pilot experiments to study the mechanism of action of InsP6 show that following treatment with InsP6, the concentration of intracellular [Ca2+] ([Ca2+]i) is increased by 57% (P less than 0.02). Likewise, a 41% increase (P less than 0.05) in InsP3 and a 26% decrease (P less than 0.02) in InsP2 were noted 1 h following treatment with InsP6. Contrary to the dogma that cell division is associated with increased [Ca2+]i, our data show that reduced cell growth and enhanced differentiation is associated with increased [Ca2+]i and increased InsP3 in the presence of InsP6.

Changes in inositol lipids and phosphates after stimulation of the MAS-transfected NG115-401L-C3 cell line by mitogenic and non-mitogenic stimuli.

A neuronal cell line (NG115-401L-C3) was stimulated by mitogenic (angiotensin) and non-mitogenic (bradykinin) peptides and examined for the time course of changes in the levels of radiolabelled inositol phosphates and phospholipids. Both peptides stimulated the time-dependent production of Ins(1,4,5)P3 and related metabolites. Bradykinin caused a much larger increase in Ins(1,4,5)P3 than did angiotensin. However, both peptides stimulated similar rises in the levels of Ins(1,3,4)P3 and InsP4. Bradykinin, but not angiotensin, caused a rapid (within 2 s) fall in the levels of PtdIns(4,5)P2 and PtdIns(4)P. Serum pretreatment of the cells caused a 2-3-fold potentiation of both the responses to bradykinin and angiotensin. Although significant levels of PtdIns(3)P were detected in resting cells, neither mitogenic (angiotensin, insulin-like growth factor I, transforming growth factor beta) nor non-mitogenic (bradykinin, nerve growth factor, interleukin-1) receptor activation changed its levels, arguing against regulation of either PtdIns 3-kinase or PtdIns(3)P phosphatase. We conclude that, as judged by the levels of its product. PtdIns(3)P, the enzyme PtdIns 3-kinase is not activated. This questions the significance of this activity in the receptor-mediated initiation of DNA synthesis.

[Substrate specificity of glycogen synthase from rabbit skeletal muscles]. / O substratnoi spetsifichnosti glikogensintazy skeletnykh myshts krolika.

Nitrous bases were shown to play an essential role in the specificity of active and adenyl nucleotide binding sites. Pyrimidine base determines the substrate specificity of rabbit skeletal muscle glycogen synthase; a crucial role in this process is ascribed to the lactam fragment of the pyrimidine cycle. The 2-oxo group was also shown to be involved in substrate binding. The adenyl nucleotide binding site interacts only with 6-aminopurine derivatives. A negative interaction was found between the enzyme active center and the adenyl nucleotide binding site.

Plasma membrane purification from roots of sunflower by phase partitioning.

Plasma membrane vesicles were purified from the roots of sunflower (Helianthus annuus L. cv. Topflor) by aqueous polymer two-phase partitioning. The optimal conditions for separation were determined by systematic variation of the polymer concentration and salt composition. The phase system containing 6% (w/w) dextran T-500, 6% (w/w) polyethylene glycol 3350, 250 mM sucrose, 5 mM potassium phosphate, pH 7.8, without added salts proved to be the best. The ATPase activity had a pH optimum at 6.5 and it was stimulated by Mg2+, but not by Ca2+. The plasma membrane MgATPase activity was inhibited by vanadate but not by nitrate, an inhibitor of tonoplast ATPase. Only 10% of the microsomal protein was responsible for 36% of the total MgATPase activity. Moreover IDPase activity, a Golgi marker, appeared to be very low indicating the high purity of the preparation.

Characterization of inositol 1,4,5-trisphosphate-stimulated calcium release from rat cerebellar microsomal fractions. Comparison with [3H]inositol 1,4,5-trisphosphate binding.

The abilities of D-myo-inositol phosphates (InsPs) to promote Ca2+ release and to compete for D-myo-[3H]-inositol 1,4,5-trisphosphate [( 3H]Ins(1,4,5)P3) binding were examined with microsomal preparations from rat cerebellum. Of the seven InsPs examined, only Ins(1,4,5)P3, Ins(2,4,5)P3 and Ins(4,5)P2 stimulated the release of Ca2+. Ca2+ release was maximal in 4-6 s and was followed by a rapid re-accumulation of Ca2+ into the Ins(1,4,5)P3-sensitive compartment after Ins(1,4,5)P3, but not after Ins(2,4,5)P3 or Ins(4,5)P2. Ca2+ re-accumulation after Ins(1,4,5)P3 was also faster than after pulse additions of Ca2+, and coincided with the metabolism of [3H]Ins(1,4,5)P3. These data suggest that Ins(1,4,5)P3-induced Ca2+ release and the accompanying decrease in intraluminal Ca2+ stimulate the Ca2+ pump associated with the Ins(1,4,5)P3-sensitive compartment. That this effect was observed only after Ins(1,4,5)P3 may reflect differences in either the metabolic rates of the various InsPs or an effect of the Ins(1,4,5)P3 metabolite Ins(1,3,4,5)P4 to stimulate refilling of the Ins(1,4,5)P3-sensitive store. InsP-induced Ca2+ release was concentration-dependent, with EC50 values (concn. giving half-maximal release) of 60, 800 and 6500 nM for Ins(1,4,5)P3, Ins(2,4,5)P3 and Ins(4,5)P2 respectively. Ins(1,4,5)P3, Ins(2,4,5)P3 and Ins(4,5)P2 also competed for [3H]Ins(1,4,5)P3 binding, with respective IC50 values (concn. giving 50% inhibition) of 100, 850 and 13,000 nM. Comparison of the EC50 and IC50 values yielded a significant correlation (r = 0.991). These data provide evidence of an association between the [3H]Ins(1,4,5)P3-binding site and the receptor mediating Ins(1,4,5)P3-induced Ca2+ release.

Characterization of nucleoside diphosphatase in the onion root tip. III. Solubilization and activation of membrane-bound enzyme(s).

UNLABELLED: The latency of nucleoside diphosphatase (NDPase) in onions root homogenates has been examined by comparing the activation of NDPase activity resulting from detergent treatment with that due to storage of homogenates for several days in the cold. Both detergent treatment and cold storage activated NDPase approximately two-fold. In both cases this activation was paralleled by the loss of enzyme activity from the membrane fractions and its appearance in the supernatants. Electrophoresis of these supernatants revealed an identifical isoenzyme pattern of 5 NDPase bands for both preparations. Enzyme kinetic studies demonstrated that NDPase from the detergent-treated homogenate and the homogenate stored in the cold as well as NDPase from the membrane and supernatant fractions from each of the homogenates all had the same Km value. These data suggest that latency of NDPase is the result of a breakdown of cellular membranes and subsequent release of NDPase. ABBREVIATIONS: DOC, deoxycholate; NDPase, nucleoside diphosphatase; IDP, inosine 5'-diphosphate; UDP, uridine 5'-diphosphate; GDP, guanosine 5'-diphosphate.

The gliovascular interphase in irradiated brain tissue. Electron microscopic investigations on location of specific phosphatases.

In the brains of rats exposed to a dose of 800 rads of gammma radiation the location of phosphatases hydrolysing the following phosphate esters: adenosinetriphosphate (ATP), cytidinetriphosphate (CTP), guanosinetriphosphate (GTP) and inosinediphosphate (IDP) was determined in the ultramicroscopic structures. Changes were observed in their location in the gliovascular interphase. Early after the exposure (6 hours) the activity of specific phosphatases decreased in brain capillaries, then (48 hours) the reaction product disappeared from the basal membrane of the capillaries and appeared in the endothelium. Later (72 hours -- 7 days) the intensity of the reaction increased in the processes of the astrocytes adhering to the capillary. These changes took place concomitantly with an increase in the permeability of the blood-brain barrier and suggested that specific phosphatases participate in the barrier mechanisms.