Splitting Of Adenosine Triphosphate Research Articles

Electrical and biochemical properties of an enzyme model of the sodium pump.

The electrochemical properties of a widely accepted six-step reaction scheme for the Na+, K+-ATPase have been studied by computer simulation. Rate coefficients were chosen to fit the nonvectorial biochemical data for the isolated enzyme and a current-voltage (I-V) relation consistent with physiological observations was obtained with voltage dependence restricted to one (but not both) of the two translocational steps. The vectorial properties resulting from these choices were consistent with physiological activation of the electrogenic sodium pump by intracellular and extracellular sodium (Na+) and potassium (K+) ions. The model exhibited K+/K+ exchange but little Na+/Na+ exchange unless the energy available from the splitting of adenosine triphosphate (ATP) was reduced, mimicking the behavior seen in squid giant axon. The vectorial ionic activation curves were voltage dependent, resulting in large shifts in apparent Km's with depolarization. At potentials more negative than the equilibrium or reversal potential transport was greatly diminished unless the free energy of ATP splitting was reduced. While the pump reversal potential is at least 100 mV hyperpolarized relative to the resting potential of most cells, the voltage-dependent distribution of intermediate forms of the enzyme allows the possibility of considerable slope conductance of the pump I-V relation in the physiological range of membrane potentials. Some of the vectorial properties of an electrogenic sodium pump appear to be inescapable consequences of the nonvectorial properties of the isolated enzyme. Future application of this approach should allow rigorous quantitative testing of interpretative ideas concerning the mechanism and stoichiometry of the sodium pump.

Anaerobic energy metabolism during severe hypoxia in the lungless salamander Desmognathus fuscus (Plethodontidae)

In the lungless salamander Desmognathus fuscus, mean body weight 4.5 g, the changes in total body concentration of adenosine triphosphate (ATP), creatine phosphate (CP) and lactace (LA) were measured during exposure to a severely hypotoxic atmosphere (P O 2 = 25 Torr) for 48 h at 13°C. ATP and CP decreased, reaching a minimum at 3 h of exposure, and LA increased, attaining maximum values after 12 to 24 h of hypoxia. Thereafter recovery was observed and control values of ATP, CP and LA were reached after 48 h of sustained hypoxia. This behavior is attributed to a biochemical adjustement to hypoxia of the metabolic machinery which leads to normalization of chemical energy stores in spite of O 2 uptake being persistently reduced to 30% of its normoxic level. The anaerobic energy yield derived from splitting of ATP and CP and from LA formation corresponded to about 2 3 of the oxidative energy deficit during the first 3 h of hypoxia. Thereafter anaerobic mechanisms were responsible for insignificant contributions to the energy balance.

Untersuchungen zur Lokalisation von Glycerophosphatase- und Nucleosidtriphosphatase-Aktivität in Siebzellen von Larix

The presence of comparatively high concentrations of ATP in sieve elements and the known energy-dependency of the transport of assimilates force to search for possible sites of energy dissipation within sieve cells and sieve tubes. One experimental approach is to look for the sites of enzymatic splitting of nucleoside triphosphates within these cells themselves. While P-protein associated ATP'ase has been found by some authors in sieve tubes of angiosperms almost no information is available for the sieve cells of gymnosperms which seem to lack typical P-protein. In the present investigation, therefore, the enzymatic splitting of glycerophosphate (GP), adenosine triphosphate (ATP), and uridine triphosphate (UTP) in the sieve cells of the secondary phloem of branches from adult trees of Larix leptolepis ( Sieb. et Zucc. ) Gord. has been studied cytochemically throughout the vegetation period. Cytochemical tests were performed with the Gomori lead salt method at pH 5.0 and with a modified Wachstein-Meisel method at pH 7.2. The effect of the non-enzymatic hydrolysis of the substrates, of unspecific lead or leadphosphate adsorption, and of fixatives on the cytochemical enzyme localization has been paid special attention. At acid pH, fully differentiated sieve cells show remarkably increased rate of splitting of both, GP and the nucleoside triphosphates. Addition of NaF (10 mM) to the substrate containing media inhibits almost completly positive enzyme localization. Only with ATP as substrate, NaF inhibition is incomplete. Addition of ouabain (1 mM) showed no inhibition. Fixation of sections in formalin or glutaraldehyde containing solutions caused considerable loss of staining, particularly in case of ATP and UTP as substrates, although the localization remained the same. Enzymatic activity was clearly localized at such structures of the sieve cell protoplast which are enriched at the ends of sieve cells and at sieve areas. At neutral pH, no such enzyme localization is obtained with GP, nor with UTP or ATP. ATP, however, now causes strong staining at the plasmalemma site. Because increased splitting of GP, ATP and UTP is only observed in fully differentiated sieve cells it is considered to be of functional significance.

Distribution and properties of an adenosine triphosphatase in the tanycyte ependyma of the IIIrd ventricle of the rat.

The distribution, histochemical properties and ultrastructural localization of adenosine triphosphate (ATP) hydrolyzing enzymes in the tanycyte ependyma of the third ventricle have been studied in female Wistar rats. Using a calcium-cobalt procedure and a lead capture technique, splitting of ATP could be demonstrated in perikarya and processes of tanycytes in the region of the ventromedial nucleus. The reaction showed no dependence on magnesium or sodium ions, did not occur with other monodi-, and tri-phosphates as substrates, and was inhibited by p-chlormercuribenzoate (PCMB) and sodium fluoride, but not by ouabain. With the calcium-cobalt method the highest intensity of reaction was found at pH 9.4, whereas the lead method gave optimal results at pH 6--8. At the ultrastructural level, the reaction product was found at the outer surface of the plasma membranes of tanycytes and reached its highest concentrations in the region of the region of the apical microvilli; From the findings it is concluded that splitting of ATP in tanycytes is due to a true ATPase. The enzyme might be involved in an active transport of substances by tanycytes.

On splitting of adenosine triphosphate by the Z-plates of indirect flight muscles from Phormia regina (Diptera)

ATP splitting is demonstrated to occur in the Z discs of isolated flight muscle myofibrils ofPhormia regina after the quantitative extraction of myosin.

INFLUENCES OF THYROXINE AND INSULIN ON THE TURNOVER AND FATE OF HIGH-ENERGY PHOSPHATE.

Ingestion by the weanling rat of thyroxine in an amount greatly in excess of physiological requirements resulted in significant increases in the rates of turnover of the high-energy compounds phosphocreatine (PC), adenosine diphosphate (ADP), and adenosine triphosphate (ATP) and in significant decreases in their concentrations in skeletal muscle. Nucleoside phosphorylase and myokinase activities and rates of pyrophosphate splitting of ATP and of utilization of ATP for nuclear syntheses were unaltered in thyrotoxic muscle. The findings suggest that increased extramitochondrial utilization of ATP for glycolysis may be the factor responsible for the increased phosphorylating capacity of the thyrotoxic animal. An effect of thyroxine on mitochondrial permeability as well as on rate of formation of the high-energy intermediate would appear to explain the observed results.A further enhancement of the already markedly elevated thyrotoxic rate of phosphorylation was effected by the superimposition of insulin. Insulin also effected increases in the concentrations of the high-energy compounds. It is apparent that insulin acts by reducing the utilization of ATP for phosphorylation of glucose and thus increases the mitochondrial availability of high-energy acceptor. This increased mitochondrial affinity for high-energy compounds results in an enhancement of mitochondrial energy transfer processes.The findings appear to involve both thyroxine and insulin in the control of cell structure.

Mitochondrial formation and hydrolysis of adenosine triphosphate in the presence of some rare-earth ions

Investigations were made to determine whether the toxic effect of rare earths and the changes in liver lipid metabolism are due to an alteration in adenosine triphosphate (ATP) metabolism in mitochondria. In order to test the possibility that oxidative phosphorylation is impaired by the parenteral administration of lanthanide metal ions prior to manifestation of fatty infiltration of the liver, the metabolism of rat-liver mitochondria was studied 8 hr after the intravenous injection of LaCl/sub 3/ or Pr(NO/sub 3/)/sub 3/. However, the P/O ratios of the liver mitochondria isolated after such pretreatment were found to be normal. The effects of the rare earth ions on non- enzymic hydrolysis of ATP at pH 7.5 to 7.5 were also tested. The data show that the trivalent ions of the rare earths tested do not stimulate the enzymic splitting of ATP by mitochondrial digitonin fragments. The data presented then do not support the view that the ions of La, Pr, Nd, and Sm have a direct toxic effect on the synthesis or breakdown of ATP in mitochondria. (P.C.H.)

Investigation of the effect of calcium ions on the splitting of adenosinetriphosphate by myosin

A kinetic analysis of the effects of Ca on myosin ATP-ase demonstrates that in the presence of 0.1 M NaCl at pH 6.5 with Ca concentrations below 10 −2 M the data can be explained in 2 different ways: either CaATP is the substrate and free ATP a competitive inhibitor, while all the myosin is active, or only the Ca-myosinate is active and free ATP is the substrate, while CaATP is inactive. As the optimal activity with intermediate Ca concentrations can be accounted for quantitatively by the last explanation without assuming any other postulates, this last explanation is favored.

Die nukleosidtriphosphatase-aktivität von L-Myosin und aktomyosin in abhängigkeit von den ionalen bedingungen

1. 1. The dependence of nucleosidetriphosphatase (NTPase) activity of actomyosin and L-myosin on the ionic conditions has been investigated. 2. 2. The following holds with regard to the NTPase activity of calcium-activated L-myosin: 2.1. (a) Ca ions activate much more strongly the splitting of 6-OH-NTP by L-myosin gel and L-myosin sol than the splitting of 6-NH 2-NTP. 2.2. (b) The order in which the 6-OH-NTP is split at an increasing speed is very markedly dependent on the ionic strength. 2.3. (c) The order in which the 6-NH 2-NTP is split is independent of the ionic strength. ATP is always split more rapidly than CTP. 2.4. (d) All the NTPs are split more rapidly by L-myosin gel than by L-myosin sol. 3. 3. The following holds with regard to NTPase activity of L-myosin in the presence of magnesium ions: 3.1. (a) In the presence of Mg ++ the rates of splitting by L-myosin are low (< 0.1 μmole P × mg protein −1 × min −1). 3.2. (b) Mg ions activate the splitting of guanosine triphosphate (GTP) and inosine triphosphate (ITP) by gel and sol. The splitting of uridine triphosphate (UTP) and cytidine triphosphate (CTP) by the gel is only insignificantly altered. The splitting of both NTPs by the sol is inhibited by magnesium. The splitting of adenosine triphosphate (ATP) is always inhibited by Mg ions. 3.3. (c) The rate at which L-myosin gel and L-myosin sol split NTP decreases in the order: GTP > ITP > UTP > CTP ≳ ATP 3.4. (d) The order of the rate of splitting of NTP is independent of the ionic strength. With increasing ionic strength the rates decrease only slightly. 4. 4. In the absence of alkaline earth and in the presence of chelating agents L-myosin sol splits 6-NH 2-NTP more rapidly than 6-OH-NTP. 4.1. (a) Without addition of alkaline earth the rates for all NTPs are low. 4.2. (b) Addition of chelating agents activates the splitting of ATP and CTP very strongly and the splitting of UTP only slightly. The splitting of ITP and GTP is inhibited. 5. 5. The NTPase activity of actomyosin gel is fundamentally different from that of actomyosin sol and L-myosin preparations. 6. 6. Both in the absence of alkaline earth and in the presence of Mg and Ca, the gel splits NTP at a decreasing rate in the order ATP ≳ CTP > UTP > ITP > GTP. Splitting of all the NTPs, with the exception of ATP, is more strongly activated by 10 −3 M Mg ++ than by 10 −2 M Ca ++. EDTA inhibits the splitting of all NTP by actomyosin gel. 7. 7. When the ionic strength increases from 0.1 to 0.4 μ the NTPase activity of actomyosin gel becomes converted to the NTPase activity of actomyosin sol. 8. 8. At an ionic strength of 0.4 μ, in the presence of Mg ++, all the NTPs dissociate actomyosin. The dissociated L-myosin has the same NTPase activity as the L-myosin sol. 9. 9. When no Mg ++ is added the actomyosin sol is dissociated completely only by ATP. CTP causes a stronger partial dissociation than UTP. ITP and GTP are ineffective. 10. 10. In the absence of alkaline earth and in the presence of Ca, the ATPase and CTPase activities of actomyosin and L-myosin sol are therefore equal and the UTPase activity of both proteins is very similar. 11. 11. When no alkaline earth is added ITP, GTP and UTP are split by the undissociated actomyosin sol and the L-myosin sol at a similar low rate. 12. 12. In the presence of Ca ++, GTP is split more rapidly than ITP by actomyosin sol-NTPase, while ITP is split more rapidly than GTP by L-myosin-NTPase. 13. 13. All NTPs are split more slowly by actomyosin sol than by L-myosin sol. 14. 14. In the presence of EDTA also, ATP dissociates actomyosin completely. UTP, ITP and GTP do not bring about dissociation in EDTA-containing actomyosin sol. 15. 15. The EDTA-activation of the ATP-splitting by actomyosin sol is less than that by L-myosin sol. UTP-, ITP- and GTP-splitting by undissociated actomyosin sol is inhibited by EDTA.

The intracellular localization of adenosinetriphosphate metabolism in skeletal muscle

The metabolism of adenosinetriphosphate (ATP) in skeletal muscle can be considered from two aspects. On the one hand there are anaerobic and aerobic systems concerned with the production of the high-energy phosphate bonds of ATP, whereas on the other hand this chemical energy is converted into mechanical work in the highly specialized contractile system. These phases in ATP metabolism show precise intracellular localization. Anaerobically the ATP is produced by soluble enzyme systems in the sarcoplasm, whereas the main aerobic source of supply is to be found in the mitochondria, the large granular components of skeletal muscle (Chappell &amp; Perry 1952, 1953). The mechanism of aerobic ATP production in muscle follows the general pattern which has already been demonstrated in other tissues such as liver, but the utilization of ATP by the myofibrils is a process which is characteristic of muscle. It is generally conceded that the energy required for contraction is derived from the splitting of ATP. Within the skeletal muscle cell there are two main sites of adenosinetriphosphatase (ATPase) activity, namely, the myofibrils (Perry 1951) and the lipoprotein granules of the sarcoplasm (Perry 1952 a ). The ATPase of the granules is dominantly magnesium activated, and although the role of this enzyme in intracellular function is not at all clear, that associated with the mitochondria may be in some way concerned with the phosphorylating function of these particles. Certainly the ATPase activity of granules with oxidative activity which can be isolated from pigeon breast muscle (Chappell &amp; Perry 1953) is very similar to that of mitochondria obtained from liver.