ATPase Module Research Articles

Effect of Hormones on Sucrose Uptake and on ATPase Activity of Citrus sinensis L. Osbeck Leaves

Carbohydrate accumulation in young, fully expanded leaves of Citrus sinensis L. Osbeck is affected by the presence of the fruitlet on the shoot. Previous work gave evidence that gibberellins may be involved in this 'fruit effect'. In the present work we have studied the effect of gibberellic acid (GA3) on 14C-sucrose uptake by leaf discs and whether its action could be due to a modulation of the plasma membrane ATPase, which maintains the H+ gradient that drives H+/sucrose co-transport.The effect of GA3 on 14C-sucrose uptake depended on the osmolarity of the assay medium. At 300 mOsm a reduction in the uptake rate was observed. The inhibitory effect of the hormone disappeared after preincubating the leaf discs with para-chloromercuri-phenylsulphonic acid (PCMPS), a sulphydril binding inhibitor. ATPase activity of isolated plasma membrane vesicles was inhibited by IAA treatments, while GA3 or ABA did not affect this enzyme, even after a 3 h preincubation period. However, in the absence of a surfactant in the assay medium, GA3, together with turgor pressure, modulated plasma membrane ATPase activity, possibly through modifications of membrane permeability. The hormone effect on 14 C-sucrose uptake may involve action on the sucrose carrier.

Structure of the nuclear encoded γ subunit of CF 0CF 1 of the diatom Odontella sinensis including its presequence

Using a PCR-product as homologous probe for screening of a cDNA library of the diatom Odontella sinensis overlapping cDNA clones were obtained which showed homologies to atpC-genes of F 0F 1-ATPases from different sources. Comparison of the deduced amino acid sequence with the N-terminal sequence of the Odontella γ subunit obtained by protein sequencing, indicated that the complete 370 amino acid protein is processed to a mature protein of 315 amino acids. The 55 amino acids comprising the presequence consists of two segments, one resembling a signal sequence for cotranslational transport through ER membranes and one showing characteristics of a transit sequence for transport of proteins into chloroplasts of higher plants. This result is discussed with respect to the particular envelope structure of chromophytic plastids consisting of four membranes. The outer membrane contains ribosomes on its cytosolic surface. As in cyanobacterial γ subunits the regulatory sequence region, which is involved in thiol modulation of chloroplast ATPase of green algae and higher plants, is absent in the Odontella γ subunit.

Inhibitory effect of insulin and cytoplasmic factor(s) on brain (Na + + K +) ATPase

(Na + + K +)ATPase activity in cerebral cortex was modulated by insulin action depending on the Mg 2+ concentration. Thus, in homogenates in the presence of 1–3 mM Mg 2+, insulin stimulated the enzyme, whereas in the presence of 4–6 mM Mg 2+ inhibition was observed. Exposure of synaptosomal membranes to the soluble fraction resulted in inhibition of ATPase activity in a dose-dependent manner. The inhibitory effect of insulin was regulated by a cytoplasmic factor in a dose-dependent manner. Similar variations to those obtained with a crude synaptosomal fraction were obtained by using a partially purified ATPase. These results indicated the importance of soluble factors in the modulation of ATPase by insulin and add more evidence in support for a role of insulin as a neuromodulator.

Thiol modulation of the thylakoid ATPase. Lack of oxidation of the enzyme in the presence of [formula omitted] in vivo and a possible explanation of the physiological requirement for thiol regulation of the enzyme

(1) Illumination of Dunaliella induced an increase in the activity of CF 1-ATPase, and of fructose-1,6-bisphosphatase (FBPase), that could be assayed under standard conditions in subsequently lysed cells. The light-induced, but not the light-independent, activities could be prevented by inclusion of methyl viologen in the preillumination medium. This effect was concluded to be due to the prevention of reduction of ferredoxin by methyl viologen. (2) Markedly higher concentrations of added methyl viologen were required to prevent induction of ATPase than FBPase. Addition of 1 mM methyl viologen to the preillumination stage prior to illumination totally prevented the appearance of light-induced FBPase, whereas a concentration of 20 mM was required to prevent light-induction of ATPase completely. (3) Although methyl viologen added to intact algae in the light subsequent to achievement of steady-state light activation of ATPase and FBPase led to the inactivation of FBPase, light-induced ATPase was completely uninhibited. This effect indicates that whereas FBPase is subject to reversible modification by thiols in the light, thiol reduction of ATPase in vivo is irreversible in the light, and oxidation only occurs upon dissipation of Δ μ H + produced by darkening. (4) Stabilisation of the thiol-reduced form of CF 1 by Δ μ H + (Shahak, Y. (1985) J. Biol. Chem. 260, 1459–1464) accounts for the lower reducing pressure necessary to activate thiol-dependent ATPase, compared to FBPase. Implications for the regulation of CO 2 fixation are discussed. (5) Complete activation of CF 1-ATPase activity was induced by incubation of intact algae with dithiothreitol in the dark. It is suggested that thiol-regulation of the enzyme is necessary in vivo in order to prevent activation of an ATPase activity which would otherwise inevitably result from autocatalytic generation of Δ μ H + in the dark.

Increase in plasma potassium concentration following indomethacin administration: absence of a role for membrane Na-K ATPase.

To elucidate whether indomethacin-induced hyperkalaemia is due to an inhibition of Na-K ATPase in the membranes, indomethacin (25 mg t.d.s.) was administered to 7 normal subjects for 7 days. This resulted in an increase in plasma potassium concentrations in all 7 subjects: median (range) for the entire group increased from 4.19 (3.98-4.79) mmol/l to 4.29 (4.13-4.87) mmol/l. Leucocytes prepared from these subjects prior to and after indomethacin were tested for 86Rb influx and [3H]-ouabain binding (an index of Na-K ATPase sites). Neither 86Rb influx (total, ouabain sensitive and ouabain insensitive) nor [3H]-ouabain binding changed significantly following indomethacin. We conclude that (a) indomethacin-induced hyperkalaemia is not due to alterations in potassium influx into cells and (b) the modulation of Na-K ATPase sites/activity is in leucocytes not dependent upon prostaglandins.

Thermotropic phase transitions in the plasma membrane of ram spermatozoa.

A steady-state fluorescence polarization technique, using the membrane probe 1,6-diphenyl-1,3,5-hexatriene (DPH), showed that separately detectable transitions occurred in the regions of 17, 26 and 36 degrees C in isolated preparations of ram sperm plasma membrane. An independent technique based on the temperature-related behaviour of calcium- and magnesium-activated ATPase detected a single phase transition in the region of 24 degrees C. Modulation of ATPase by neighbouring lipid composition was inferred from findings that phospholipase A2 caused significant stimulation of the enzyme. Cholesterol-rich liposomes caused an upward shift of the phase-transition temperature from 24 degrees C to 30 degrees C, but the reasons for this are unclear. It is considered that these phase transitions may have profound effects on sperm survival and physiology, both during normal fertilization processes and in response to cryostorage.

Modulation of smooth muscle actomyosin ATPase by thin filament associated proteins

Caldesmon binds equally to both gizzard actin and actin containing stoichiometric amounts of bound tropomyosin. The binding of caldesmon to actin inhibits the actin-activation of the Mg-ATPase activity of phosphorylated myosin only when the actin contains bound tropomyosin. The reversal of this inhibition requires Ca 2+-calmodulin; but it occurs without complete release of bound caldesmon. Although phosphorylation of the caldesmon occurs during the ATPase assay, a direct correlation between caldesmon phosphorylation and the release of the inhibited actomyosin ATPase is not consistently observed.

Modulation of the light-activated ATPase by background light

Modulation of the light-activated ATPase by background light

Calcium-sensitive modulation of the actomyosin ATPase by fodrin.

Fodrin, a spectrin-like protein isolated from brain, is a long flexible molecule which binds calmodulin and cross-links F-actin. The effects of fodrin on the actin-activated ATPase of myosin have been examined. When added after ATP, fodrin inhibited the actomyosin ATPase. Two to three times as much fodrin was required for inhibition in the presence of Ca2+ as in its absence. Complete inhibition in the absence of Ca2+ occurred at about one fodrin to 200 actins. Inhibition does not appear to result from fodrin cross-linking F-actin, and, thereby, preventing the myosin filaments from reaching the actin filaments; but cross-linking may promote inhibition by trapping the myosin filaments within the cross-linked F-actin. When added before ATP, fodrin stimulated the actomyosin ATPase almost 3-fold in the presence of Ca2+ and by less than 50% in the absence of Ca2+. Stimulation is thought to result from fodrin cross-linking F-actin. After several minutes the stimulations in Ca2+ were greatly reduced, and in the absence of Ca2+ the actomyosin ATPases were substantially inhibited. Whether added before or after ATP, fodrin inhibited the actin-activated ATPase of myosin subfragment 1. This inhibition was also slightly Ca2+ sensitive.

Thiol modulation of the chloroplast protonmotive ATPase and its effect on photophosphorylation

Thiol modulation of the chloroplast protonmotive ATPase (CF 0-CF 1) by preillumination of broken chloroplasts in the presence of dithiothreitol (or preillumination of intact chloroplasts in the absence of added thiols) had the following effects on photophosphorylation. (1) When assayed at pH 8 and saturating light, the initial rate of photophosphorylation was increased by 10–40%. There was an accompanying increase in the rate of coupled electron transport with no significant change in the overall P 2 e ratio. (2) On lowering the pH of the assay medium to pH 7, the stimulatory effect of thiol modulation on photophosphorylation and coupled electron flow was enhanced. At pH 7, there was also a small increase in P 2 e ratio. (3) Addition of a non-saturating amount of uncoupler to the assay medium enhanced the stimulatory effect of thiol modulation on photophosphorylation. In the presence of 1 mM NH 4Cl, there was only a small increase in coupled electron flow and a correspondingly larger increase in P 2 e ratio. (4) Lowering the light intensity, or inhibiting electron transport, diminished the stimulatory effect of thiol modulation on photophosphorylation, coupled electron transport and P 2 e ratio. (5) Under all the above conditions, the ΔpH maintained across the thylakoid membrane was lower after thiol modulation, even when photophosphorylation markedly increased in rate. (6) Thiol modulation of CF 0-CF 1 increased the observed Michaelis constant for ADP ( K m(ADP)) and the apparent maximum rate ( V app of photophosphorylation by the same factor, so that ratio V app K m was not altered. V app K m was also unaffected by changing the medium pH, but was significantly decreased upon addition of uncouplers to the medium. These results indicate that the observed rate of ATP synthesis catalysed by thiol demodulated chloroplasts is limited kinetically by the fraction (α) of enzyme molecules that are active during photophosphorylation. A model based on a dual pH optimum requirement for activation of CF 0-CF 1 is presented to explain the dependence of α on ΔpH. Thiol modulation of CF 0-CF 1 is proposed to stimulate photophosphorylation by causing the enzyme to become active over a lower range of ΔpH, thereby reducing the kinetic limitation on ATP synthesis imposed by the activation process.

Linoleate incorporation into rat liver membranes phospholipids: Effect on plasma membrane atpase activities and physical properties

Plasma membrane phospholipids were modified by incubation in the presence of linoleyl-CoA with or without added lysolecithin (LPC) for various lenghth of time. In the absence of LPC, a maximum of 10 nmoles linoleyl-phosphatidylcholine (PC) were synthesized and the ATPase specific activities were not affected whereas in the presence of LPC, when linoleyl-PC synthesis rose from 10 to 80 nmoles, the ATPase activities were decreased. The decrease was similar in the Na,K- or in the Mg-dependant-ATPase and reached maximally 30–40%. LPC by itself did not modify the ATPases. A concomitant decrease in DPH polarization was observed when linoleate was incorporated into phospholipids. We concluded that the decreased ATPase specific activities may be due to an increased fluidity of membranes produced by linoleyl-PC synthesis. We compare this modulation of ATPases by the membrane fluidity with the specific effect of linoleyl-PC species on adenylate cyclase.

Modulation of the chloroplast ATPase by tight ADP binding. Effect of uncouplers and ATP.

Inactivation of the membrane-bound ATPase by tight ADP binding was studied under nonenergized conditions. The energy state of the system was controlled either by omitting MgCl2, preventing ATP hydrolysis, or by addition of an uncoupler which dissipates the delta mu H+. In the absence of Mg2+, ATP prevents the inactivation of the enzyme by ADP, in a competitive manner. This effect of ATP resembles that of GDP with Mg2+ present. In the presence of nigericin, Mg2+, and ATP, inactivation occurs after a 10-15-sec interval, during which the enzyme is able to hydrolyze ATP at a relatively rapid rate. The degree of inactivation is proportional to the level of bound ADP detected. This behavior is different from that of the coupled ATPase (no uncoupler added), where inactivation is attained only upon exhaustion of the ATP by its hydrolysis, despite the finding that ADP binds tightly to the active ATPase at all stages of the reaction. Higher levels of tightly bound ADP were detected in the presence of an uncoupler. We suggest that the interval during which the enzyme becomes inactive is that required for the enzyme to generate and bind ADP, and to change from the active to the inactive conformation. These results support the mechanism suggested previously for the modulation of the ATPase by tight nucleotide binding.

Modulation of the chloroplast ATPase by tight binding of nucleotides

Inactivation of the chloroplast ATPase upon tight nucleotide binding was studied with several adenine nucleotide analogs. Compared with ADP, the other nucleoside diphosphates were less effective in the follwing order: IDP > ϵ-ADP > 1-oxido-ADP > GDP. The nucleotide analogs compete with ADP for binding to the tight nucleotide-binding site(s) on the ATPase and also prevent further inactivation by ADP. Ado PP[NH] P also causes inactivation but has a lower affinity than ADP. [ 3H]GDP binds tightly to the ATPase, but the resulting enzyme-GDP complex is more readily dissociable than the enzyme-ADP complex. Although both nucleotides appear to bind to the same site, the catalytic and binding properties of the coresponding nucletide-enzyme complexes differ. Binding of GDP also decreases the rate and extent of the sontaneous decay of the activated enzyme. PP i decreases the rate of inacivation caused by ADP and also the level of tigthly buond ADP. Based on these results, we suggest that two different confomations of the ATPase exist which contain tigthly bound ADP. The active conformation is conveted to the inactive conformation in the absence of a continued supply of energy by illumination or ATP hydrolysis.

Modulation of the calcium-transport ATPase in human erythrocytes by anions

Certain polyanionic agents, such as poly( l-aspartic acid) ( M r = 20 000) and poly( l-glutamic acid) ( M r = 20 000 and 26 000) were shown to increase the Ca 2+ sensitivity of (Ca 2+ + Mg 2+)-ATPase activity in erythrocytes by converting the biphasic (high and low affinity) Ca 2+ activation observed in Tris-maleate buffer to a predominantly hyperbolic Ca 2+ activation of high affinity. Aspartic acid and glutamic acid, however, were ineffective. The enzyme also exhibited a single high Ca 2+ affinity state in the presence of the alicyclic sulfonic acids, sodium Hepes (55 mM) or sodium Mes (55 mM), but not with the aliphatic sulfonic acid, sodium Tes (55 mM). This effect of both the poly( l-carboxylic acid)s and the sulfonic acid buffers did not require the presence of an intact membrane, as it was also observed in a Triton X-100 solubilized (Ca 2+ + Mg 2+)-ATPase preparation. In contrast to the polyanions and the sulfonic acids, the anionic Ca 2+ binding protein, calmodulin, increased the maximum velocity of the enzyme as well as its Ca 2+ sensitivity. Certain aromatic carboxylic acids (benzoic acid and salicyclic acid) also increased the Ca 2+ -sensitivity of the (Ca 2+ + Mg 2+)-ATPase, whereas a variety of inorganic anions tested were ineffective at 66 mM concentrations. Trifluoperazine (30 μM) antagonized activation of the enzyme by calmodulin and poly( l-asparatic acid), but not by sodium Hepes or sodium Mes. The activation by poly( l-carboxylic acid)s or sodium Hepes was not additive to a similar activation produced by trypsin. These results suggest that the modulation of the Ca 2+ sensitivity of (Ca 2+ + Mg 2+)-ATPase by calmodulin is associated with its anionic properties, and that this property can be mimicked by other anions, probably by interaction at an anion-regulatory site on the enzyme.

Myosin light chain phosphorylation is associated with a decrease in the energy cost for contraction in fast twitch mouse muscle.

The rate of splitting of energy-rich phosphate compounds and the extent of myosin light chain phosphorylation in contracting mouse extensor digitorum longus (EDL, fast twitch) and soleus (slow twitch) muscles were studied at 20 degrees C. The rate of high energy phosphate-splitting during a maintained isometric tetanus was 1.44 +/- 0.21 mumol . g-1 . s-1 in soleus. In EDL, the splitting rate was higher, 3.71 +/- 0.62 mumol . g-1 . s-1, during the first several seconds and thereafter was reduced to a rate of 1.63 +/- 0.35 mumol . g-1 . s-1 between 12 and 15 s of stimulation. Light chains identified on 2-dimensional gel electrophoretograms from EDL corresponded to the light chain composition of fast twitch muscle (LC1f, LC2f, and LC3f). Soleus is composed of fast twitch and slow twitch fibers because 2 additional light chains were found: LC1s and LC2s. In unstimulated EDL and soleus muscles, 0.1 of the LC1f and LC2s were phosphorylated. Upon stimulation, only LC2f, and only in EDL, increased its extent of phosphorylation. The time course of increase in phosphorylation of LC2f and decrease in rate of high energy phosphate-splitting correspond so that the 2 processes may be mechanistically related. If so, it appears that myosin LC2f phosphorylation represents a thick filament regulatory system capable of downward modulation of actomyosin ATPase in vivo during a maintained contraction.

Modulation of coupling factor ATPase activity in intact chloroplasts. Reversal of thiol modulation in the dark

Illumination of intact pea chloroplasts results in both the pH activation and the thiol modulation of the reversible protonmotive ATPase, or CF 0-CF 1 (Mills, J.D., Mitchell, P. and Schürmann, P. (1980) FEBS Lett. 112, 173–177). We have studied the reversal of the activation and thiol-modulation processes that occurs when illuminated chloroplasts are darkened. (1) Methyl viologen is shown to prevent light-dependent thiol modulation of CF 0-CF 1 in intact chloroplasts. Studies using methyl viologen indicate that the decline in ATPase activity of intact chloroplasts in the dark is due both to pH deactivation and thiol demodulation of CF 0-CF 1. Reillumination in the presence of methyl viologen reactivates CF 0-CF 1 complexes that have undergone pH deactivation, but does not allow thiol modulation to occur. (2) At 20°C, both pH deactivation and thiol demodulation of CF 0-CF 1 are complete within 10 min darkness. At 3.5°C, both processes are retarded, and require more than 30 min for completion. (3) Lysis of intact chloroplasts greatly retards the thiol demodulation of CF 0-CF 1. In the dark, addition of oxidants to lysed chloroplasts promotes thiol demodulation. Under these conditions, thioredoxin behaves as a weak oxidant, but glutathione has very little effect. Illumination of intact chloroplasts followed by rapid lysis and storage on ice stabilises the pH-activated, thiol-modulated state of CF 0-CF 1 for extended periods in the dark and increases the time required for thiol demodulation to well over 2 h. This method may prove to be useful for preparing thiol-modulated chloroplasts without the need to add or remove exogenous thiols. The results suggest that an oxidative system exists in the stroma which actively reverses thiol modulation of CF 0-CF 1 in the dark. Such a system would be expected if thiol modulation of CF 0-CF 1 were an important physiological process regulating reversible ATPase activity in vivo.

Temperature-pressure modulation of the (Na +K +) ATPase during low temperature acclimation in periplaneta americana

Temperature-pressure modulation of the (Na +K +) ATPase during low temperature acclimation in periplaneta americana