The binding of precursor proteins to chloroplasts requires nucleoside triphosphates in the intermembrane space.

Protein import into chloroplasts is initiated by a binding interaction between a precursor protein and the surface of the outer envelope. The binding step was previously shown to be energy-dependent (Olsen, L. J., Theg, S. M., Selman, B. R., and Keegstra, K. (1989) J. Biol. Chem. 264, 6724-6729). We took advantage of the broad nucleotide specificity of the energy requirement for binding to investigate the site of the nucleoside triphosphate (NTP) requirement. GTP supported precursor binding to chloroplasts. It was not converted to ATP, as determined by direct ATP measurements, and was not transported across the inner envelope. Thus, GTP supported binding from either the intermembrane space or outside the outer membrane. To distinguish between an intermembrane space and an external NTP requirement, we experimentally manipulated the NTP levels inside and outside chloroplasts. Internally generated ATP was able to support binding in the presence of an external membrane-impermeant ATP trap. Therefore, since GTP supported binding from either the intermembrane space or outside the chloroplast, and ATP supported binding from either the intermembrane space or the stroma, we concluded that the site of NTP utilization for precursor binding to chloroplasts was the intermembrane space between the two envelope membranes.     

Analysis of the energy metabolism after incubation of Saccharomyces cerevisiae with sulfite or nitrite

After addition of 5 mM sulfite or nitrite to glucose-metabolizing cells of Saccharomyces cerevisiae a rapid decrease of the ATP content and an inversely proportional increase in the level of inorganic phosphate was observed. The concentration of ADP shows only small and transient changes. Cells of the yeast mutant pet 936, lacking mitochondrial F₁ATPase, after addition of 5 mM sulfite or nitrite exhibit changes in ATP, ADP and inorganic phosphate very similar to those observed in wild type cells. They key enzyme of glucose degradation, glyceraldehyde-3-phosphate dehydrogenase was previously shown to be the most sulfiteor nitrite-sensitive enzyme of the glycolytic pathway. This enzyme shows the same sensitivity to sulfite or nitrite in cells of the mutant pet 936 as in wild type cells. It is concluded that the effects of sulfite or nitrite on ATP, ADP and inorganic phosphate are the result of inhibition of glyceraldehyde-3-phosphate dehydrogenase and not of inhibition of phosphorylation processes in the mitochondria. Levels of GTP, UTP and CTP show parallel changes to ATP. This is explained by the presence of very active nucleoside monophosphate kinases which cause a rapid exchange between the nucleoside phosphates. The effects of the sudden inhibition of glucose degradation by sulfite or nitrite on levels of ATP, ADP and inorganic phosphate are discussed in terms of the theory of Lynen (1942) on compensating phosphorylation and dephosphorylation in steady state glucose metabolizing yeast.Abbreviations ATP adenosine triphosphate - ADP adenosine diphosphate - AMP adenosine monophosphate - P_i inorganic orthophosphate Dedicated to Prof. Dr. Hans Grisebach on the occasion of his sixtieth birthday     

Cardiac sarcolemmal and sarcoplasmic reticulum membrane vesicles exhibit distinctive (Ca-Mg)-ATPase substrate specificities

The nucleoside 5'-triphosphate (NTP) substrate specificities for Ca-stimulated ATPase and ATP-dependent Ca2+ uptake activities have been examined in cardiac sarcolemma (SL) and sarcoplasmic (SR) membrane vesicles. The results indicate that SL membrane vesicles exhibit a much narrower range of NTP substrate specificities than SR membranes. In SR membrane vesicles, the Ca-stimulated Mg-dependent hydrolysis of ATP and dATP occurred at nearly equivalent rates, whereas the rates of hydrolysis of GTP, ITP, CTP, and UTP ranged from 16-33% of that for ATP. All of the above nucleotides also supported Ca2+ transport into SR vesicles; dATP was somewhat more effective than ATP while GTP, ITP, CTP, and UTP ranged from 28-30% of the activity for ATP. In the presence of oxalate, the initial rate of Ca accumulation with dATP was 4-fold higher than for ATP, whereas the activity for GTP, ITP, CTP, and UTP ranged from 35-45% of that for ATP. For the SL membranes, Ca-activated dATP hydrolysis occurred at 60% of the rate for ATP; GTP, ITP, CTP, and UTP were hydrolyzed by the SL preparations at only 7-9% of the rate for ATP. NTP-dependent Ca2+ uptake in SL membranes was supported only by ATP and dATP, with dATP 60% as effective as ATP. GTP, ITP, CTP, and UTP did not support the transport of Ca2+ by SL vesicles. The results indicate that the SL and SR membranes contain distinctly different ATP-dependent Ca2+ transport systems.     

Effect of nucleotide cofactor structure on recA protein-promoted DNA pairing. 1. Three-strand exchange reaction.

The structurally related nucleoside triphosphates, adenosine triphosphate (ATP), purine riboside triphosphate (PTP), inosine triphosphate (ITP), and guanosine triphosphate (GTP), are all hydrolyzed by the recA protein with the same turnover number (17.5 min-1). The S0.5 values for these nucleotides increase progressively in the order ATP (45 microM), PTP (100 microM), ITP (300 microM), and GTP (750 microM). PTP, ITP, and GTP are each competitive inhibitors of recA protein-catalyzed ssDNA-dependent ATP hydrolysis, indicating that these nucleotides all compete for the same catalytic site on the recA protein. Despite these similarities, ATP and PTP function as cofactors for the recA protein-promoted three-strand exchange reaction, whereas ITP and GTP are inactive as cofactors. The strand exchange activity of the various nucleotides correlates directly with their ability to support the isomerization of the recA protein to a strand exchange-active conformational state. The mechanistic deficiency of ITP and GTP appears to arise as a consequence of the hydrolysis of these nucleotides to the corresponding nucleoside diphosphates, IDP and GDP. We speculate the nucleoside triphosphates with S0.5 values greater than 100 microM will be intrinsically unable to sustain the strand exchange-active conformational state of the recA protein during ongoing NTP hydrolysis and will therefore be inactive as cofactors for the strand exchange reaction.     

Kinetics of the acclimational responses of tench to combined hypoxia and hypercapnia

Summary Exposing tench to environmental hypoxia-hypercapnia reduces routine O₂ consumption, sharply decreases arterial O₂ tension and the difference between the water and the blood, and results in marked swelling of the erythrocytes. These changes are rapidly reversed upon return to normoxia.Hypoxic-hypercapnic conditions lower the blood NTP/Hb ratio to a new steady state level within 24 h, by reducing GTP/Hb but not ATP/Hb. A similar selective reduction of eryhtrocytic GTP content forms the initial response of blood incubated in vitro to anoxic conditions.The swelling as well as the reduced GTP/Hb ratio in the erythrocytes appear to improve O₂ loading in the gills during environmental hypoxia-hypercapnia.Symbols and abbreviations a arterial - GTP guanosine triphosphate - Hct hematocrit - I inspired - NTP nucleoside triphosphate - w water     

Vesicular preparation of a highly coupled ATPase-ATP synthase complex from pig heart mitochondria

1. A method is described to prepare an ATPase-ATP synthase complex from pig heart mitochondria exhibiting a very high ATP-32Pi exchange activity (1.6 mumol/min per mag protein in optimal conditions). 2. The preparation is virtually devoid of nucleoside diphosphokinase and adenylate kinase activities. 3. Freeze-fracture studies show that the ATPase-ATP synthase complex is integrated in lipid vesicles of 400-600 A in diameter. 4. It contains the endogenous natural proteic inhibitor which seems to behave as a coupling factor. 5. The rate of ATP hydrolysis catalyzed by the ATPase-ATP synthase complex is competitively inhibited by ADP, while the presence of ADP increases the initial rate of 32Pi incorporation into ATP. 6. The 32Pi incorporation into ATP can occur at a rate almost equal to that of nucleoside triphosphate (NTP) hydrolysis provided that the rate of NTP hydrolysis is kept low and that the ADP concentration is high enough. In these conditions, a very high coupling between NTP hydrolysis and ATP synthesis can be demonstrated.     

Analysis of teleost hemoglobin by Adair and Monod-Wyman-Changeux models

Summary The allosteric effects of the erythrocytic nucleoside triphosphates (NTP) and of proton concentrations were investigated by precise measurement of Hb–O₂ equilibria of tench hemoglobin (including extreme, high and low saturation ranges) and analysed in terms of the MWC two state model and the Adair four step oxygenation theory.At low concentrations (NTP/Hb ratio=1.0, and pH>7.3) ATP, GTP and protons decrease Hb–O₂ affinity by increasing the allosteric constantL and reducingK _T, the association constant¹ of the deoxy, tense state of the Hb, without significantly affecting that (K _R) of the oxy state, increasing the free energy of cooperativity (G). High concentrations of these effectors, however, also reduceK _R. The greater sensitivity of the half-saturation O₂ tension (P ₅₀) of the Hb to GTP than to ATP at the same concentration, correlates with greater effects of GTP on bothK _T andK _R. The pH and NTP dependence of the four Adair association constants and the calculated fractional populations of Hb molecules in different stages of oxygenation show that the autochthonous NTP effectors and protons stabilize the T structure and postpone the TR transition basic to cooperativity in fish Hb.The possible implications of the findings for aquatic respiration are discussed.Abbreviations ATP adenosine triphosphate - DPG 2,3-diphosphoglycerate (glycerate-2,3-bisphosphate) - GTP guanosine triphosphate - IHP inositol hexaphosphate - NTP nucleoside triphosphates In this paperK _T andK _R are defined as theassociation equilibrium constants instead of dissociation constants (as originally defined by Monod et al. 1965) to facilitate comparison with the Adair constants     

The role of nucleoside-diphosphate kinase reactions in G protein activation of NADPH oxidase by guanine and adenine nucleotides

NADPH-oxidase-catalyzed superoxide (O2-) formation in membranes of HL-60 leukemic cells was activated by arachidonic acid in the presence of Mg2+ and HL-60 cytosol. The GTP analogues, guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S] and guanosine 5'-[beta,gamma-imido]triphosphate, being potent activators of guanine-nucleotide-binding proteins (G proteins), stimulated O2- formation up to 3.5-fold. The adenine analogue of GTP[gamma S], adenosine 5'-[gamma-thio]triphosphate (ATP[gamma S]), which can serve as donor of thiophosphoryl groups in kinase-mediated reactions, stimulated O2- formation up to 2.5-fold, whereas the non-phosphorylating adenosine 5'-[beta,gamma-imido]triphosphate was inactive. The effect of ATP[gamma S] was half-maximal at a concentration of 2 microM, was observed in the absence of added GDP and occurred with a lag period two times longer than the one with GTP[gamma S]. HL-60 membranes exhibited nucleoside-diphosphate kinase activity, catalyzing the thiophosphorylation of GDP to GTP[gamma S] by ATP[gamma S]. GTP[gamma S] formation was half-maximal at a concentration of 3-4 microM ATP[gamma S] and was suppressed by removal of GDP by creatine kinase/creatine phosphate (CK/CP). The stimulatory effect of ATP[gamma S] on O2- formation was abolished by the nucleoside-diphosphate kinase inhibitor UDP. Mg2+ chelation with EDTA and removal of endogenous GDP by CK/CP abolished NADPH oxidase activation by ATP[gamma S] and considerably diminished stimulation by GTP[gamma S]. GTP[gamma S] also served as a thiophosphoryl group donor to GDP, with an even higher efficiency than ATP[gamma S]. Transthiophosphorylation of GDP to GTP[gamma S] was only partially inhibited by CK/CP. Our results suggest that NADPH oxidase is regulated by a G protein, which may be activated either by exchange of bound GDP by guanosine triphosphate or by thiophosphoryl group transfer to endogenous GDP by nucleoside-diphosphate kinase.     

Effect of nucleotide cofactor structure on recA protein-promoted DNA pairing. 2. DNA renaturation reaction.

We have examined the effects of the structurally related nucleoside triphosphates, adenosine triphosphate (ATP), purine riboside triphosphate (PTP), inosine triphosphate (ITP), and guanosine triphosphate (GTP), on the recA protein-promoted DNA renaturation reaction (phi X DNA). In the absence of nucleotide cofactor, the recA protein first converts the complementary single strands into unit-length duplex DNA and other relatively small paired DNA species; these initial products are then slowly converted into more complex multipaired network DNA products. ATP and PTP stimulate the conversion of initial product DNA into network DNA, whereas ITP and GTP completely suppress network DNA formation. The formation of network DNA is also inhibited by all four of the corresponding nucleoside diphosphates, ADP, PDP, IDP, and GDP. Those nucleotides which stimulate the formation of network DNA are found to enhance the formation of large recA-ssDNA aggregates, whereas those which inhibit network DNA formation cause the dissociation of these nucleoprotein aggregates. These results not only implicate the nucleoprotein aggregates as intermediates in the formation of network DNA, but also establish the functional equivalency of ITP and GTP with the nucleoside diphosphates. Additional experiments indicate that the net effect of ITP and GTP on the DNA renaturation reaction is dominated by the corresponding nucleoside diphosphates, IDP and GDP, that are generated by the NTP hydrolysis activity of the recA protein.     

Regulatory properties of magnesium-dependent guanylate cyclase in Dictyostelium discoideum membranes

We have characterized a magnesium-dependent guanylate cyclase in homogenates of Dictyostelium discoideum cells. 1) The enzyme shows an up to 4-fold higher cGMP synthesis in the presence of GTP analogues with half-maximal activation at about 1 microM guanosine 5'-O-(3-thio)triphosphate (GTP gamma S) or 100 microM guanosine 5'-(beta, gamma-imido)triphosphate; little or no stimulation was observed with GTP, guanosine mono- and diphosphates or with adenine nucleotides, with the exception of the ATP analogue adenosine 5'-(beta, gamma-imido)triphosphate. 2) Both basal and GTP gamma S-stimulated guanylate cyclase activity were rapidly lost from homogenates as was the ability of GTP gamma S to stimulate the enzyme after cell lysis. 3) Inclusion of 25 microM GTP gamma S during cell lysis reduced the KM for GTP from 340 to 85 microM and increased the Vmax from 120 to 255 pmol/min.mg protein, as assayed in homogenates 90 s after cell lysis. 4) Besides acting as an activator, GTP gamma S was also a substrate for the enzyme with a KM = 120 microM and a Vmax = 115 pmol/min.mg protein. 5) GTP gamma S-stimulated, Mg2+-dependent guanylate cyclase was inhibited by submicromolar concentrations of Ca2+ ions, and by inositol 1,4,5-trisphosphate in the absence of Ca2+ chelators. 6) Guanylate cyclase activity was detected in both supernatant and pellet fractions after 1 min centrifugation at 10,000 x g; however, only sedimentable enzyme was stimulated by GTP gamma S. We suggest that the Mg2+-dependent guanylate cyclase identified represents the enzyme that in intact cells is regulated via cell surface receptors, and we propose that guanine nucleotides are allosteric activators of this enzyme and that Ca2+ ions play a role in the maintenance of the enzyme in its basal state.     

Comparison between ATP-supported and GTP-supported phosphate turnover of the calcium-transporting sarcoplasmic reticulum membranes.

The study deals with the interrelationship of the phosphate-transferring activities of the calcium-transporting sarcoplasmic reticulum membrane vesicles: the phosphate exchange between nucleoside triphosphate (NTP) and nucleoside diphosphate (NDP) (NTP-NDP exchange), the calcium-dependent NTase, and the phosphorylation of NDP by inorganic phosphate in the presence of NTP (NTP-Pi exchange). Different nucleotides were used as phosphate donors and acceptors. It is demonstrated for the phosphate transfer from ITP to GDP that the NTP-NDP exchange exhibits ping-pong kinetics with Mg-ITP and unliganded GDP as substrates. The apparent affinities of the enzyme for the nucleoside diphosphate and triphosphate species are deduced according to this mechanism. The enzyme's affinity for the nucleoside triphosphates and diphosphates depends on its functional state being considerably lower under conditions of NTP-NDP exchange than during NTP splitting or NTP synthesis. ATP and GTP are split with the same low rates when calcium-activated NTPase is inhibited by high internal calcium concentrations after calcium transport has reached steady state. The rates of the NTP-NDP exchange reactions, however, differ by a factor of about 10 being approximately equal to 3 mumol . mg-1 . min-1 for ATP-ADP and only approximately equal to 0.3 mumol . mg-1 . min-1 (22 degrees C) for GTP-GDP. When the sarcoplasmic reticulum vesicles are made calcium-permeable, the calcium transport ATPase is turned on and the rates of GTP and ATP splitting increase about tenfold. Yet, while the rate of ATP-ADP exchange is little reduced, the rate of GTP-GDP exchange drops by approximately 50%. The persisting exchange activity of calcium-permeable vesicles demonstrates that high internal calcium concentrations are not required for the transfer of the protein-bound phosphoryl group to NDP during NTP-NDP exchange.     

Changes in blood metabolites following stress from capture and handling of the marine teleost Girella tricuspidata

Whole blood nucleoside triphosphate (NTP) and lactate in the parore (Girella tricuspidata, Fam: Kyphosidae) were monitored over a period of 12 hr following capture by gill net. An increase in NTP during the post-capture recovery period was mainly attributable to a significant rise (P less than 0.05) in the NTP component guanosine triphosphate (GTP). The rise in GTP levels correlated with the decline in blood lactate (r = -0.72) accumulated during the period of capture stress. It is suggested that metabolism of lactate via the Krebs cycle may be responsible for the rise in GTP.     

Characterization of a GDP-sensitive phosphorylation in plasma membranes of D. discoideum

In a previous study, we reported the GDP-dependent phosphorylation of a 36 kD membrane protein, p36, inD. discoideum membranes prepared from starved (aggregation competent) cells (Anschutzet al., 1989). Here we show that p36 can be phosphorylated when membranes are supplied either ATP or GTP as the phosphate donor, but that a greater level of p36 phosphorylation is achieved with GTP. The rate of phosphorylation of p36, using either nucleotide triphosphate, is enhanced by GDP. This reflects a decrease in the apparentK _m of the enzyme for the particular nucleotide triphosphate. p36 can also be phosphorylated in membranes prepared from vegetative cells. However, the ability of GDP to stimulate p36 phosphorylation is not observed in vegetative cell membranes. Competition experiments indicate that there are also developmental differences in the nucleotide triphosphate site(s) available to phosphorylate p36.     

Modulation of nucleotide specificity of thermophilic F(o)F(1)-ATP Synthase by epsilon-subunit

The C-terminal two α-helices of the ε-subunit of thermophilic Bacillus F(o)F(1)-ATP synthase (TF(o)F(1)) adopt two conformations: an extended long arm ("up-state") and a retracted hairpin ("down-state"). As ATP becomes poor, ε changes the conformation from the down-state to the up-state and suppresses further ATP hydrolysis. Using TF(o)F(1) expressed in Escherichia coli, we compared TF(o)F(1) with up- and down-state ε in the NTP (ATP, GTP, UTP, and CTP) synthesis reactions. TF(o)F(1) with the up-state ε was achieved by inclusion of hexokinase in the assay and TF(o)F(1) with the down-state ε was represented by εΔc-TF(o)F(1), in which ε lacks C-terminal helices and hence cannot adopt the up-state under any conditions. The results indicate that TF(o)F(1) with the down-state ε synthesizes GTP at the same rate of ATP, whereas TF(o)F(1) with the up-state ε synthesizes GTP at a half-rate. Though rates are slow, TF(o)F(1) with the down-state ε even catalyzes UTP and CTP synthesis. Authentic TF(o)F(1) from Bacillus cells also synthesizes ATP and GTP at the same rate in the presence of adenosine 5'-(β,γ-imino)triphosphate (AMP-PNP), an ATP analogue that has been known to stabilize the down-state. NTP hydrolysis and NTP-driven proton pumping activity of εΔc-TF(o)F(1) suggests similar modulation of nucleotide specificity in NTP hydrolysis. Thus, depending on its conformation, ε-subunit modulates substrate specificity of TF(o)F(1).     

Adenylate gradients and Ar:O2 effects on legume nodules. II. Changes in the subcellular adenylate pools

Central infected zone tissue of soybean (Glycine max L. Merr.) nodules was fractionated into separate subcellular compartments using density gradient centrifugation in nonaqueous solvents to better understand how exposure to Ar:O(2) (80:20%, v/v) atmosphere affects C and N metabolism, and to explore a potential role for adenylates in regulating O(2) diffusion. When nodules were switched from air to Ar:O(2), adenylate energy charge (AEC) in the plant cytosol rose from 0.63 +/- 0.02 to 0.73 +/- 0.02 within 7 min and to 0.80 +/- 0.01 by 60 min. In contrast, AEC of the mitochondrial compartment of this central zone tissue remained high (0.80 +/- 0.02 to 0.81 +/- 0.02) following Ar treatment while that of the bacteroid compartment was unchanged, at 0.73 +/- 0.02, after 7 min, but declined to 0.57 +/- 0.03 after 60 min. These results were consistent with a simulation model that predicted Ar:O(2) exposure would first reduce ATP demand for ammonia assimilation and rapidly increase cytosolic AEC, before the Ar:O(2)-induced decline mediated by a decrease in nodule O(2) permeability reduces bacteroid AEC. The possibility that adenylates play a key, integrating role in regulating nodule permeability to oxygen diffusion is discussed.     

Control of protein synthesis in Escherichia coli: Lack of correlation with changes in intracellular pools of ATP,GTP, and ppGpp

Intracellular pools of ATP, GTP, and ppGpp have been measured in Escherichia coli after an energy source shift-down from glucose minimal to succinate-minimal medium. In a Tic⁺ strain (ATCC 10798), which reduces translational initiation after the down-shift, the rate of protein labeling falls to about 30% of its preshift rate within the first minute after shift and reaches a minimum of 17% by 6 min after shift. The ATP pool in this strain remains constant for about 10 min after shift, then declines gradually to about 60% of its initial level. The temporal discrepancy between protein synthesis and the decline in the ATP pool indicates that a decrease in intracellular ATP is not necessary for the control of protein synthesis. In a Tic^? strain (W1), which cannot control translational initiation under these conditions, the decline in the ATP pool is somewhat more rapid and more pronounced (to 40%) than in the Tic⁺ strain, indicating that the decline in the ATP pool is not sufficient to trigger control of translational initiation. The intracellular GTP pool in the Tic⁺ strain remains constant for 2 min after shift, then declines gradually to reach a minimum of 45% of its initial level at 20 min after shift. The pattern is in general similar in the the Tic^? strain, although the ultimate decline in GTP is more pronounced (to 29%). These data indicate that the decline in GTP is not sufficient and probably unnecessary to elicit control of translational initiation. Intracellular levels of ppGpp increase with very similar kinetics in relA⁺Tic⁺ (ATCC 10798) and relA⁺Tic^? (W1) strains, indicating that elevated ppGpp levels are not sufficient to elicit control of translation. In a relA^?Tic⁺ strain (NF162), or in a relA⁺Tic⁺ strain treated with rifampin, the ppGpp pool does not increase significantly after shift-down although translational initiation is reduced. Thus, an increase in the ppGpp pool is not necessary to control of translational initiation.     

Time-Resolved Analysis of Signals Involved in Systemic Induction of Pin2 Gene Expression

The systemic induction of proteinase inhibitor genes in tomato plants is mediated either by electrical signals, hydraulic signals or chemical messengers. In the present study we analyzed the effects of mechanical wounding, heat treatment and electrical current application on wild-type tomato plants (Lycopersicon esculentum Mill, cv Moneymaker) and ABA-deficient mutants of tomato (sitiens). Kinetic studies revealed that systemic Pin2 gene expression could be slightly induced by the fast transient membrane potential change which left the damaged leaf within 30–60s after wounding. Moreover, a signal leaving the damaged tissue between 2 and 4 minutes after wounding was responsible for a significant amplification of Pin2 gene expression. This signal could either be a decrease in turgor pressure, which occurred 3–4min after treatment, or a slow electrical transient. In addition, mechanical wounding and electrical current seem to involve ABA to induce changes in membrane potential and to promote Pin2 gene expression. In contrast, heat triggers fast and slow electrical transients leading to an induction of Pin2 gene expression within the plant independently of ABA. Turgor pressure, in turn, is presumably adjusted in relation to ionic movements across the membrane, elucidated by membrane potential recordings. In conclusion, wound-induced changes in membrane potential seem to be dependent on the endogenous level of ABA. These shifts in membrane potentials, in turn, are involved in regulation of turgor pressure within the plant.     

Nucleotide Phosphohydrolase in Purified Vaccinia Virus

Purified infectious vaccinia virus has been shown to contain an enzyme or enzymes that remove the terminal phosphate group from adenosine triphosphate (ATP), guanosine triphosphate (GTP), uridine triphosphate (UTP), and cytidine triphosphate (CTP). The K(m) for ATP of this enzyme is 5.5 x 10(-4)m, and the relative rates of the reaction with ATP, GTP, UTP, and CTP are 1.00, 0.34, 0.15, and 0.29, respectively. The virus enzyme does not react with any of the diphosphates. The rate of the reaction is proportional to the amount of virus added and is linear for 130 min. The virus nucleotide phosphohydrolase activity is greatly stimulated by Mg(++) and very slightly stimulated by Ca(++). The small residual activity observed in the absence of divalent cations is completely inhibited by ethylenediaminetetraacetic acid. Neither Na(+) nor K(+) ions, nor any mixture of these, was found to stimulate the reaction significantly, and ouabain, at 10(-4)m, inhibited the reaction by only 27%. The response of the vaccinia enzyme to mono- and divalent cations and to ouabain indicates that the vaccinia enzyme has different properties from those associated with microsomes and mitochondria.     

Effects of experimental anaemia on intra-erythrocytic phosphate levels in rainbow trout, Oncorhynchus mykiss

The concentrations of intra-erythrocytic adenylates (ATP, ADP and AMP) and guanylates (GTP, GDP and GMP) were determined in rainbow trout subjected to 10% blood removal every 12 h for 96 h. Haemoglobin concentration, [Hb], decreased from 6·043±0·617 to 0·957 ± 0·195 g dl⁻¹. This decrease in [Hb] was followed by a continuous increase in total organic phosphates, e.g. adenylates plus guanylates. Intra-erythrocytic NTP (ATP plus GTP) levels increased significantly after 48 h when haemoglobin concentration was 2·427 ± 0·256 g dl⁻¹. Although a significant increase in GDP levels in animals with [Hb] less than 1·677 ± 0·235 g dl⁻¹ was observed, the general increase in guanylate level was mainly due to the GMP which increased about 85-fold during the experimental period. It is suggested that the erythrocytes of anaemic rainbow trout have the capacity to increase NTP/Hb₄ ratios which may represent an advantage for anaemic fish.