Adenosine triphosphatase of bean plastids: its properties and site of formation

Extracts of bean (Phaseolus vulgaris L.) etioplasts and chloroplasts contain a dithiothreitol-activated Ca²⁺-dependent adenosine triphosphatase which is inhibited by Dio-9. The chloroplast and etioplast enzymes have identical R_F values upon disc gel electrophoresis. Optimum extraction of the enzyme from either plastid preparation is accomplished with 1 mm ethylenediamine tetraacetic acid. Photophosphorylation capacity can be partially restored to depleted chloroplast preparations by addition of either the chloroplast or etioplast extract. These results suggest that the adenosine triphosphatase from etioplasts and chloroplasts represents a modified coupling factor for photophosphorylation.     

Adenosine triphosphate-lead histochemical reactions in ependymal epithelia of murine brains do not represent calcium transport adenosine triphosphatase

Summary The strong enzyme histochemical reactions for adenosine triphosphatase (ATPase) seen in ependymal tanycytes after incubation in calcium-containing media have previously been reported as calcium transport ATPase. Investigation of these reactions showed that: (1) any nucleoside triphosphate can serve as a substrate; (2) diphosphates and monophosphates cannot replace triphosphates; this includes p-nitrophenyl phosphate which is readily hydrolysed by plasma membrane transport ATPases; (3) strong localization occurs in the presence of millimolar concentrations of either calcium or magnesium ions; there is no absolute requirement for calcium ions; (4) they are not inhibited by sulphydryl inhibitors or calmodulin antagonists; (5) lead phosphate precipitates are localized almost entirely on the external face of tanycyte plasma membranes. In addition, the technique gives strong localization to vessels in the choroid plexus but not to the choroidal epithelium. Immunohistochemistry with a primary antibody raised against Ca²⁺,Mg²⁺-ATPase stains the choroidal epithelium but not the vessels or the ependymal tanycytes. These results are inconsistent with identification of the reaction as calcium transport ATPase but support characterization as an ecto-ATPase.     

Redistribution of phosphate deposits in the algaScenedesmus quadricauda deprived of exogenous phosphate—an ultra-cytochemical study

Summary The green algaScenedesmus quadricauda (Turp.) Bréb. was cultivated in the presence or absence of orthophosphate and synchronized daughter or mother cells were cytochemically stained. Forin situ capturing of water soluble phosphates Ca²⁺ and Mg²⁺ ions were added to the ice-cold glutaraldehyde fixative to form a polymeric metal-phosphate complex which was equivalent to the energy-rich condensed polyphosphates in staining by alkaline lead acetate. The X-ray microanalysis of the extensive stained deposits proved the presence of phosphorus. In orthophosphate-supplied daughter cells cytoplasmic vacuoles contained round stained bodies; a layer of phosphate-containing paracrystals encompassing some starch grains and a fine stained layer delineating the chloroplast envelope were also observed. In the equivalent mother cells only the material inside theloculi of stacked thylakoids was stained. In orthophosphate starved daughter cells filamentous phosphate-containing paracrystals filled extensive cytoplasmic vacuoles. A stained layer covered the chloroplast envelope and continuous stained layers appeared inside theloculi of stacked thylakoids. Mother cells that develop from these daughter cells were filled with starch grains and showed only peripheral stained deposits. The results are compared with the biochemical evidence of phosphate turnover in algal cells.Abbreviations ADP adenosine diphosphate - ATP adenosine triphosphate - ATPase adenosine triphosphatase - EDAX energy dispersive analysis of X-rays - Pi orthophosphate - PPi pyrophosphate - PP polyphosphate - PhAR photosynthetic active radiation - TCA trichloroacetic acid     

Characterization of an ATPase Associated with the Inner Envelope Membrane of Amyloplasts from Suspension-Cultured Cells of Sycamore (Acer pseudoplatanus L.)

Amyloplast envelope membranes isolated from cultured, white-wild cells of sycamore (Acer pseudoplatanus L.) have been found to contain a Mg²⁺-ATPase, ranging in specific activity from 5 to 30 nanomoles per minute per milligram protein. This ATPase hydrolyzes a broad range of nucleoside triphosphates, whereas it hydrolyzes nucleoside mono- and diphosphates poorly, if at all. The ATPase activity was stimulated by several divalent cations, including Mg²⁺, Mn²⁺ and Ca²⁺, whereas it was not affected by Sr²⁺, K⁺, or Na⁺. The K_m for total ATP was 0.6 millimolar, and the activity showed a broad pH optimum between 7.5 and 8.0. The ATPase was insensitive to N,N′-dicyclohexylcarbodiimide and oligomycin, but it was inhibited by vanadate. All these characteristics are basically similar to those reported previously for the Mg²⁺-ATPase of the chloroplast inner-envelope membrane. Likewise, the amyloplast envelope enzyme was shown to be located specifically on the inner envelope membrane. The amyloplast envelope membranes were chemically modified with a series of unique affinity labeling reagents, the adenosine polyphosphopyridoxals (M Tagaya, T Fukui 1986 Biochemistry 25: 2958-2964). About 90% of the ATPase activity was lost when the envelope membranes were preincubated with 0.1 millimolar adenosine triphosphopyridoxal. Notably, the enzyme was protected completely from inactivation in the presence of its substrate, ATP. In contrast, both adenosine diphosphopyridoxal and pyridoxal phosphate caused much less of an inhibitory effect. This greater relative reactivity of the triphosphopyridoxal analog is similar to that reported previously with Escherichia coli F₁ ATPase (T Noumi et al. 1987 J Biol Chem 262: 7686-7692).     

Adenosine triphosphatase activity associated with mung bean mitochondria

The properties of the adenosine triphosphatase activity associated with tightly coupled, time-stable mung bean (Phaseolus aureus Roxb.) mitochondria resemble those of intact animal mitochondria. Induction of adenosine triphosphatase activity by 2,4-dinitrophenol was inhibited by oligomycin, oxidizable substrates, and high concentrations of sucrose. Upon sonication, high rates of endogenous adenosine triphosphate hydrolysis resulted, an absolute requirement for Mg²⁺ was manifested, stimulation by 2,4-dinitrophenol and inhibition by sucrose were eliminated, but sensitivity to oligomycin was retained.     

Properties of a Partially Purified Nucleoside Triphosphatase (NTPase) from the Chloroplast Envelope of Pea

The Mg-nucleoside triphosphatase activity associated with the inner envelope membrane of the pea chloroplast is comprised of at least two components, a major activity that is sensitive to vanadate and sodium fluoride and a minor insensitive activity. The vanadate/fluoride sensitive activity has been partially purified (about 35-fold) from Triton X-100 solubilized membranes by DEAE-Sephadex chromatography and sucrose density gradient centrifugation. The partially purified enzyme resembles the membrane-bound activity in requiring either Mg²⁺ or Mn²⁺, having a broad specificity for nucleoside triphosphates, having a K_m for ATP of 0.18 millimolar, and being inhibited by N-ethylmaleimide, but insensitive to sodium azide and dicyclohexylcarbodiimide. The partially purified enzyme obtained after sucrose gradient centrifugation has a markedly increased sensitivity to inhibition by inorganic pyrophosphate compared with the less pure enzyme. Pyrophosphate is not a substrate of either the membrane-bound or partially purified enzyme.     

Transport adenosine triphosphatase activity in the rat cornea

Summary The sodium-potassium activated adenosine triphosphatase (NaKATPase) activity of the rat cornea was investigated histochemically using a Pb²⁺-precipitation technique in which adenosine triphosphate (ATP) is used as substrate and two methods for potassium-dependent para-nitrophenyl-phosphatase (K-NPPase) activity.With all the three techniques used it was demonstrated that the sodium-potassium-activated adenosine triphosphatase (NaK-ATPase) activity is localized in the cell membranes of the endothelium whereas a much weaker activity was observed in the epithelium. When the Pb²⁺-technique was used, the epithelial cell membranes showed a weaker reaction in the presence of ouabain. This activity was only Mg²⁺-dependent and was presumably due to an Mg²⁺-dependent ATPase.The validity of the histochemical techniques for NaK-ATPase activity is discussed. The results emphasize the importance of the endothelium as the main site of Na⁺ transport in the cornea. Small amounts of the enzyme are also present in the epithelium, which seems to be rich in Mg²⁺-ATPase. Provided that careful controls are performed, all the methods give consistent results in the cornea.The work is part of an eye research project by Arto Palkama and supported by grants from the Sigrid Jusélius Foundation, Helsinki, Finland, to A.P. and from the Finnish Cultural Foundation, Helsinki, Finland, to T.T. and M.P.The authors are grateful to Miss Irma Hiltunen for skilful technical assistance     

The action of thiol reagents on the adenosine-triphosphatase activities of heavy meromyosin and L-myosin

1. The Ca²⁺-activated adenosine triphosphatase of heavy meromyosin is maximally stimulated by lower relative molar concentrations of phenylmercuric acetate than are required with myosin. 2. Stimulation of the Ca²⁺-activated adenosine triphosphatase of both heavy meromyosin and myosin by thiol reagents is markedly affected by ionic strength, the effects being greater with the former than with the latter. In particular, N-ethylmaleimide strongly inhibits the Ca²⁺-activated adenosine triphosphatase of heavy meromyosin at ionic strength below about 0·2. 3. The precise behaviour of the thiol reagents at low ionic strength is slightly modified by the age of the heavy meromyosin and myosin preparations. 4. Stimulation of the Mg²⁺-activated adenosine triphosphatase of heavy meromyosin by thiol reagents is relatively insensitive to ionic strength. 5. The adenosine triphosphatases of heavy meromyosin and myosin activated by potassium chloride in the absence of bivalent activators are inhibited by thiol reagents over the range of ionic strength at which stimulation occurs in the presence of calcium chloride as activator. 6. The modifying effects of potassium chloride and sodium chloride are qualitatively different when heavy-meromyosin adenosine triphosphatase is stimulated with phenylmercuric acetate. No such difference is observed when the enzyme is stimulated with N-ethylmaleimide.     

Reconstitution of the energy-linked transhydrogenase activity in membranes from a mutant strain of Escherichia coli K12 lacking magnesium ion- or calcium ion-stimulated adenosine triphosphatase

1. We have isolated a mutant of Escherichia coli K12 (strain AN295) that forms de-repressed amounts of Mg²⁺,Ca²⁺-stimulated adenosine triphosphatase. 2. The Mg²⁺,Ca²⁺-stimulated triphosphatase activity was separated from membrane preparations from strain AN295 by extraction with 5mm-Tris–HCl buffer containing EDTA and dithiothreitol, resulting in a loss of the ATP-dependent transhydrogenase activity. The non-energy-linked transhydrogenase activity remained in the membrane residue. 3. The solubilized Mg²⁺,Ca²⁺-stimulated adenosine triphosphatase activity from strain AN295 was partially purified by repeated gel filtration. The addition of the purified Mg²⁺,Ca²⁺-stimulated adenosine triphosphatase to the membrane residue from strain AN295 reactivated the ATP-dependent transhydrogenase activity. 4. Strain AN296, lacking Mg²⁺,Ca²⁺-stimulated adenosine triphosphatase activity, was derived by transducing the mutant allele, uncA401, into strain AN295. The ATP-dependent transhydrogenase activity was lost but the non-energy linked transhydrogenase was retained. 5. The ATP-dependent transhydrogenase activity in membrane preparations from strain AN296 (uncA⁻) could not be re-activated by the purified Mg²⁺,Ca²⁺-stimulated adenosine triphosphatase from strain AN295. However, after extraction by 5mm-Tris–HCl buffer containing EDTA and dithiothreitol, the ATP-dependent transhydrogenase activity could be re-activated by the addition of the purified Mg²⁺,Ca²⁺-stimulated adenosine triphosphatase from strain AN295 to the membrane residue from strain AN296 (uncA⁻).     

Phosphatases in the central nervous system of spiders

Summary The central nervous systems of web-building spiders (Araneidae, Agelenidae) and hunting spiders (Lycosidase, Salticidae) were tested for non-specific and specific phosphatases. Acid phosphatase exhibited weakly to moderately positive reactions in the neuronal cell bodies and in the neuropile fibre mass of all species investigated. Alkaline phosphatase could only be demonstrated in the external and internal neural lamellae of the brain and ventral cord of several specimens of the araneid species investigated. Tests for thiamine pyrophosphatase were negative with both the lead and calciumcobalt methods. Distinctive positive reactions for adenosine triphosphatase were visible in the nervous system of all the species used, being especially strong in the optic ganglia of the hunting spiders. The demonstration of adenosine triphosphatase was only possible when applying the calcium-cobalt method after Padykula and Herman, while the lead method after Wachstein and Meisel did not produce any staining reaction at all. Controls of the histochemical reaction showed that the enzyme was activated by Ca²⁺ and inhibited by sulphydryl destroying reagents (e.g. PCMB), but was insensitive to ouabain. It could be probably classified as a mitochondrial proton-translocating adenosine triphosphatase.     

K stimulation of ATPase activity associated with the chloroplast inner envelope

Studies were conducted to characterize ATPase activity associated with purified chloroplast inner envelope preparations from spinach (Spinacea oleracea L.) plants. Comparison of free Mg²⁺ and Mg·ATP complex effects on ATPase activity revealed that any Mg²⁺ stimulation of activity was likely a function of the use of the Mg·ATP complex as a substrate by the enzyme; free Mg²⁺ may be inhibitory. In contrast, a marked (one- to twofold) stimulation of ATPase activity was noted in the presence of K⁺. This stimulation had a pH optimum of approximately pH 8.0, the same pH optimum found for enzyme activity in the absence of K⁺. K⁺ stimulation of enzyme activity did not follow simple Michaelis-Menton kinetics. Rather, K⁺ effects were consistent with a negative cooperativity-type binding of the cation to the enzyme, with the K_m increasing at increasing substrate. Of the total ATPase activity associated with the chloroplast inner envelope, the K⁺-stimulated component was most sensitive to the inhibitors oligomycin and vanadate. It was concluded that K⁺ effects on this chloroplast envelope ATPase were similar to this cation's effects on other transport ATPases (such as the plasmalemma H⁺-ATPase). Such ATPases are thought to be indirectly involved in active K⁺ uptake, which can be facilitated by ATPase-dependent generation of an electrical driving force. Thus, K⁺ effects on the chloroplast enzyme in vitro were found to be consistent with the hypothesized role of this envelope ATPase in facilitating active cation transport in vivo.     

Adenylate kinase bound to the envelope membranes of spinach chloroplasts.

The activity of adenylate kinase (ATP:AMP phosphotransferase, EC 2.7.4.3) in both the forward (2ADP → ATP + AMP) and backward (ATP + AMP → 2ADP) reactions was found to be associated with the envelope membranes which were isolated from spinach chloroplasts. Sonication and repeated washing in a medium of high ionic strength were unable to release the enzymes from the envelope membranes. Adenylate kinase bound to the envelope is stable in the cold and inactivated by heat and acid treatments. The enzyme requires magnesium ion as an activator. The pH-activity profile of the forward reaction catalyzed by membrane-bound adenylate kinase gave a maximal activity at pH 8.5. The apparent Michaelis constant, K_m, value for ADP in the forward reaction was estimated to be 1.3 ± 0.2 × 10^?4m. A Lineweaver-Burk plot of the forward reaction gave a straight line when the reciprocal of the reaction rate was plotted versus the reciprocal, and not the square of the reciprocal, of the concentration of substrate ADP. This favors the view that the adenylate kinase bound to the chloroplast envelope has a single or equivalent binding site of Mg-ADP^?. The probable involvement of adenylate kinase bound to the chloroplast envelope in controlling the energy pool and adenylate translocation in chloroplasts is suggested.     

Relative deficiency of Ca2 plus-dependent adenosine triphosphatase activity of red cell membranes in hereditary spherocytosis

The Ca²⁺-dependent adenosine triphosphatase activity associated with the plasma membrane of normal human erythrocytes is similar to that of erythrocytes from patients with hereditary spherocytosis. When spherocytic ghosts are compared to age-matched controls, however, they show a significantly decreased Ca²⁺-dependent adenosine triphosphatase activity. The role of the relative deficiency of Ca²⁺-dependent adenosine triphosphatase in spherocytic ghosts is discussed in the light of the effects of intracellular [Ca²⁺] on the deformability and the rigidity of the cell membrane. This enzyme may be involved in the molecular mechanism of hereditary spherocytosis.     

Development and use of chlorotetracycline fluorescence as a measurement assay of chloroplast envelope-bound mg

Experiments were conducted to develop chlorotetracycline (CTC) fluorescence as an assay of Mg²⁺ bound to the envelope of the intact chloroplast. This assay technique has been widely used to measure envelope associated divalent cations in animal cell and subcellular systems, but has not been used with chloroplasts. Chloroplast envelope-associated Mg²⁺ was altered by pretreatment with Mg²⁺ and divalent cation chelating agents and by additions of Mg²⁺ to the CTC assay medium. Results indicated that for a given chloroplast preparation, relative changes in envelope-associated Mg²⁺ can be effectively monitored with CTC fluorescence. It was concluded that the limitations of this assay system are: (a) chlorophyll strongly quenches CTC fluorescence signal, so a constant chlorophyll concentration must be maintained, (b) measurements must be made quickly, and (c) use of the technique to compare different chloroplast preparations may not be valid. Studies with ²⁸Mg²⁺ confirmed our interpretation of the fluorescence results, and also suggested that the chloroplast envelope is fairly impermeable to Mg²⁺. It was concluded that changes in Mg²⁺ associated with the chloroplast due to incubation of plastids in solutions containing up to 5 millimolar Mg²⁺ may be exclusively due to increased envelope-associated Mg²⁺. The CTC assay was used in experiments to demonstrate that increases in chloroplast envelope-associated Mg²⁺ inhibit photosynthetic capacity. This inhibition can be partially overcome by the presence of K⁺ in the photosynthetic reaction media.     

THYMIDINE TRIPHOSPHATE SYNTHESIS IN TETRAHYMENA : I. Studies on Thymidine Kinase

The amount of thymidine-H³ converted to thymidine-H³ monophosphate in 30 min formed the basis for assays of thymidine kinase in cell extracts from Tetrahymena pyriformis. The optimal concentration of adenosine triphosphate is lower than that required by other cell types. Thymidine triphosphate does not exercise any feedback control of the enzyme. Other deoxyprimidine nucleotides were tested, but these also failed to exhibit any feedback inhibition. At suboptimal adenosine triphosphate levels, thymidine triphosphate and other deoxypyrimidine nucleotides stimulate the reaction, suggesting that these nucleotides may act either directly or indirectly as phosphate donors in the crude enzyme preparations. This possibility was affirmed when thymidine triphosphate and deoxycytidine triphosphate were shown to be capable of limited phosphorylation of thymidine. Comparison of enzymatic activities in logarithmically growing culture and stationary phase culture, in which nuclear DNA synthesis has virtually ceased, reveals no change in enzymatic activity. The results suggest that thymidine kinase is a constitutive enzyme in Tetrahymena.     

The localization and properties of membrane adenosine triphosphatases in the guinea-pig placenta

Summary The distribution and properties of cytochemically demonstrable phosphatases in the near-term guinea-pig placenta were examined using a strontium capture technique for sodium- and potassium-dependent adenosine triphosphatase (Na⁺, K⁺-ATPase) and a lead capture technique for magnesium-dependent adenosine triphosphatase (Mg²⁺-ATPase).Localizations with the strontium technique in the presence of an alkaline phosphatase inhibitor were mainly on the syncytiotrophoblast plasma membranes; the reaction was potassium-dependent and ouabain-sensitive. Reaction product using the lead capture method was found on both trophoblast and endothelial cell plasma membranes and was independent of magnesium and insensitive to p-hydroxymercuribenzoate (POHMB), an inhibitor of membrane ATPases. However, a very large proportion of this reaction could be blocked by an alkaline phosphatase inhibitor.It is concluded that the strontium capture technique gave a reliable localization for Na⁺, K⁺-ATPase. However, the lead capture method mainly demonstrated alkaline phosphatase, and does not offer a useful approach to specific ATPase studies in this particular system.     

Purification and characterization of myo-inositol hexaphosphate-adenosine diphosphate phosphotransferase from Phaseolus aureus

myo-Inositol hexaphosphate adenosine diphosphate phosphotransferase transfers phosphate from myo-inositol hexaphosphate to adenosine diphosphate to synthesize adenosine triphosphate. This enzyme has been isolated and purified from ungerminated mungbean seeds and found to be different from guanosine diphosphate phosphotransferase. A purification of about 200-fold with 15% recovery has been obtained. The optimal pH of the reaction is 7.0 and is dependent on the presence of a divalent cation, i.e., Mg²⁺ and Mn²⁺. The K_m value for myo-inositol hexaphosphate has been found to be 0.41 × 10^?4m and V is 90.0 nmol of P_i transferred per milligram of protein per 20 min. K_m for ADP is 0.88 × 10^-4m and V is 83.3 nmol of phosphorus transferred to ADP per milligram of protein per 20 min. The ADP phosphotransferase reaction is reversible to the extent of about 50% of the forward reaction. dADP is partly effective as an acceptor but other ribonucleoside mono- and diphosphates cannot substitute for ADP. The products ATP and myo-inositol pentaphosphate have been confirmed by several criteria. It has also been shown that this enzyme transfers phosphate only from a specific phosphoryl group (C-2 position) of myo-inositol hexaphosphate for the synthesis of ATP and 1,3,4,5,6-myo-inositol pentaphosphate or pentakis (dihydrogen phosphate).     

Increased Membrane-bound Adenosine Triphosphatase Activity Accompanying Development of Enhanced Solute Uptake in Washed Corn Root Tissue

Washing of excised corn (Zea mays L., variety WF9×M14) root tissue is accompanied by an increase in (Mg²⁺ + K⁺)-stimulated adenosine triphosphatase. This is the adenosine triphosphatase described by Fisher, Hansen, and Hodges as positively correlated with ion accumulation rates. The increase in activity is confined to the microsomal fraction. A close parallel exists between increases in adenosine triphosphatase and phosphate absorption, and they respond similarly to inhibitors of RNA and protein synthesis. However, the amplitude of change is much smaller in adenosine triphosphatase. Possible reasons for this discrepancy are discussed.     

Influence of nucleotides, cations and nucleoside triphosphatase inhibitors on the release of ribonucleic acid from isolated rat liver nuclei.

The reasons underlying reported discrepancies in the effects of ATP, ADP, adenosine 5'-[beta gamma-methylene]triphosphate, AMP + PPi, P-chloromercuribenzoate and F- on RNA efflux from isolated rat liver nuclei and on nuclear envelope nucleoside triphosphatase activity were investigated. The stimulatory effect of ADP was attributed to myokinase activity associated with the nuclei; this activity was eluted on repeated washing with nuclear incubation medium. In the absence of Ca2+ and Mn2+, ATP, adenosine 5'[beta gamma-methylene]triphosphate and AMP +PPi were found to promote release of both DNA and RNA. In the presence of 0.5 mM-Ca2+ and 9.3 mM-Mn2+, only ATP promoted RNA efflux to a significant extent. In the absence of spermidine, Ca2+ and Mn2+, nuclei released large quantities of DNA and RNA into the medium; this effect was promoted by p-chloromereuribenzoate. In the presence of the three cations, however, p-chloromercuribenzoate inhibited RNA efflux. F- caused a slight leakage of DNA from nuclei. The results are discussed in terms of models for the effects of ATP and analogues on RNA efflux and nuclear stability.     

Influence of temperature acclimatization on the ionic activation of goldfish intestinal adenosine triphosphatase

1. An adenosine triphosphatase membrane system, dependent on Mg²⁺ and activated further by Na⁺+K⁺, was prepared from goldfish anterior intestine by differential centrifugation of homogenized intestinal scrapings. 2. The affinity of this preparation for Na⁺ in the presence of K⁺+Mg²⁺, for K⁺ in the presence of Na⁺+Mg²⁺ and for Mg²⁺ alone, measured at 37°, did not depend on the previous environmental temperature of the fish. When Na⁺+K⁺ were added to preparations from 8°-acclimatized fish the affinity for Mg²⁺ increased; this was not seen with preparations from 30°-acclimatized fish. 3. Part of the Mg²⁺-activated adenosine triphosphatase was inhibited by Na⁺ and the amount of inhibition appeared to increase at high acclimatization temperatures. 4. This Na⁺-inhibited adenosine triphosphatase was separated from the (Na⁺+K⁺)-activated enzyme by centrifugation on sucrose density gradients. 5. Preparations from 8°-acclimatized fish contained less Mg²⁺-activated and more (Na⁺+K⁺)-activated adenosine triphosphatase than did similar fractions from 30°-acclimatized fish. 6. Acclimatization to different environmental temperatures might involve one form of adenosine triphosphatase changing to another. The origin of various membranes seen in microsomal fractions must, however, be established before this hypothesis can be tested further.