A study of sodium release in the course of ATP hydrolysis by membrane ATPase

Summary With the aid of sodium-sensitive glass electrodes, changes in sodium ion activity were studied in the course of subsequent additions of components required for ATP hydrolysis provided by Na⁺–K⁺-dependent membrane ATPase. Membrane ATPase was obtained from guinea pig kidney cortex. In the presence of ATP, Mg⁺⁺ and Na⁺ in media, the addition of K⁺ caused an increase in Na⁺ activity. The omission of ATP or its substitution by ADP as well as the addition of Ca⁺⁺ to the media eliminated the above-mentioned increase of Na⁺ activity. Quabain did not affect Na⁺ release caused by the addition of K⁺, although it significantly inhibited ATPase activity of the preparation. The data obtained were considered to be a direct indication of ion exchange during the course of membrane ATPase reaction. This ion-exchange stage of the reaction is not inhibited by ouabain. The ratio of sodium ions released per one inorganic phosphate formed in the course of the reaction was found to be much higher than that established for transporting membranes of intact cells. A possible cause of this difference is discussed.     

Light and electron microscopic investigation of ATPase activity in musculature during anuran tail resorption

Summary The histochemical activity of adenosine triphosphatase (ATPase) was studied at light and electron microscopic levels in larval tail musculature of Rana catesbeiana and Rana ornativentris during late metamorphic stages. The presence of low, moderate or dark reaction of K₂-EDTA-preincubated Ca⁺⁺-ATPase was correlated with the variable degree of degeneration of white fibres even at the late stage of tail resorption. The reasons for an increase in this ATPase activity in degenerating white muscle fibres are discussed. Irrespective of the degree of degeneration, all red fibres showed high ATPase reaction. During myocytolysis, it is shown that the SR vesicles accumulate electron dense amorphous material. The degree of myofibrillar disintegration correlated with decrease in ultrastructural reaction product for Mg⁺⁺-ATPase. Although grouped atrophy of muscle fibres (as seen in Xenopus laevis, den Hartog Jager et al., 1973, 1975) was absent in musculature of resorptive tails, ultrastructural characteristics including proliferation of SR and dilation of its vesicles represent alteration of the normal neural influence on the skeletal muscle fibres.     

Methodische Studien zur Zytochemie der Leukozytenphosphatasen

Zusammenfassung Die Technik des Nachweises der a. L.-Ph. in Blutausstrichen wird besprochen. Zur Fixierung wird die Methode vonKaplow empfohlen: 90% Methanol, 10% Formol 1:10, 30 sec bei 0°C. Vergleichswerte mit anderen Fixantien werden aufgeführt.Metallionen aktivieren die a. L.-Ph. in derart fixierten Ausstrichen in der Reihenfolge abnehmender Wirksamkeit:Mg⁺⁺-Fe⁺⁺⁺-Co⁺⁺-Mn⁺⁺-Cu⁺⁺-Fe⁺⁺. Die Wirkung aller Ionen erwies sich als stark konzentrationsabhängig. Ni⁺⁺, Zn⁺⁺ und Pb⁺⁺ hemmten ebenfalls konzentrationsabhängig.In den Blutausstrichen ist die Spaltungsgeschwindigkeit des sauren Na--Naphthylphosphats (Azo-Kupplung) weit höher als die des -Glycerophosphats bei maximaler Mg⁺⁺-Aktivierung mit der Calcium-Kobalt-Methode nachGomori-Takamatsu. Die Spaltungsgeschwindigkeit von -Glycerophosphat kann durch Zusetzen kleiner Mengen Fe⁺⁺⁺ und Cu⁺⁺ über die Mg⁺⁺-Aktivierung hinaus gesteigert werden bei Beschleunigung der Anfangsgeschwindigkeit der Hydrolyse.

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Summary A technic is described for demonstrating the activity of alkaline phosphatase in human leucocytes. For fixation the method ofKaplow is recommended: 90 per cent methanol +10 per cent formalin 1:10 for 30 seconds at 0°C. Other fixatives are evaluated.Under the conditions of the experiment metal ions activated the alkaline phosphatase of leucocytes in the following order: Mg⁺⁺>Fe⁺⁺⁺>Co⁺⁺>Mn⁺⁺>Cu⁺⁺>Fe⁺⁺.The effectiveness of all these metal ions was to a high degree dependent on concentration. Ni⁺⁺, Zn⁺⁺, and Pb⁺⁺ showed an inhibitory effect also dependent on concentration.In fixed blood smears the velocity of hydrolysis of sodium--naphthylphosphate (simultaneous azo-coupling technic) is far greater than that of Na--glycerophosphate (Ca-Co-method of Gomori-Takamatsu). Adding small amounts of Fe⁺⁺⁺ and Cu⁺⁺ to the incubation medium, it is possible to increase the velocity of hydrolysis of glycerophosphate beyond the values of maximal Mg⁺⁺ activation and simultaneously enhancing the initial velocity of the reaction.

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Mit 2 Textabbildungen     

New ATP incorporating activities from rat liver mitochondria: the attachment of a portion of ATP to a pronase-sensitive receptor

Studies using a Brij 58 detergent extract of rat liver mitochondria reveal that these organelles can catalyze the time-dependent incorporation of a portion of [³H]ATP into an acid-insoluble product. The activities studied using 8 mM Mn⁺⁺ or 15 mM Mg⁺⁺ are stimulated by dithiothreitol and by CTP, GTP or UTP, while that studied using 2 mM Mg⁺⁺ is not. The incorporated tritium remains bound after incubation in the presence of excess unlabeled ATP and chromatography on Sephadex G-25. The labeled product is insensitive to ribonuclease A and snake venom phosphodiesterase, but is sensitive to pronase. The attached portion of the ATP molecule released upon treatment of the product has been tentatively identified as adenosine for the activities studied using 2 mM Mg⁺⁺ or 8 mM Mn⁺⁺ and as AMP (80%) and adenosine (20%) for the reaction studied using 15 mM Mg⁺⁺.     

Improved localization of thiamine pyrophosphatase activity in mounted cryostat sections using gel fixation and gel incubation media

Summary The presence of 1% agar in the fixation and substrate solutions for the histochemical demonstration of thiamine pyrophosphatase (4.4 mM TPP; 3.6 mM Pb²⁺; 0.025 Tris-maleate buffer, pH 7.2) clearly facilitates the localization of the enzyme in Golgi apparatus in cold microtome sections prepared from unfixed specimens.     

Effects of conditions favoring enzyme phosphorylation and dephosphorylation on the activity of the alpha-keto acid dehydrogenases, with particular reference to the branched-chain alpha-keto acid dehydrogenase activities.

These studies have shown that in the crude system of rat liver mitochondria the branched-chain α-keto acid dehydrogenase activities are activated at high (10.0mM) Mg⁺⁺ concentrations favoring dephosphorylation, and are inactive at low (1.0mM) Mg⁺⁺ concentrations favoring phosphorylation. In this crude system, α-Ketoglutarate dehydrogenase activity was also regulated in this manner. In general, the optimum Mg⁺⁺ and ATP levels for activation were 10mM and 1.0mM respectively.     

Active Uptake of Ca++ and Ca++-Activated Mg++ ATPase in Red Cell Membrane Fragments

Isolated human red blood cell membrane fragments (RBCMF) were found to take up Ca⁺⁺ in the presence of ATP.¹ This ATP-dependent Ca⁺⁺ uptake by RBCMF appears to be the manifestation of an active Ca⁺⁺ transport mechanism in the red cell membrane reported previously (Schatzmann, 1966; Lee and Shin, 1969). The influences of altering experimental conditions on Ca⁺⁺-stimulated Mg⁺⁺ ATPase (Ca⁺⁺ ATPase) and Ca⁺⁺ uptake of RBCMF were studied. It was found that pretreatment of RBCMF at 50°C abolished both Ca⁺⁺ ATPase and Ca⁺⁺ uptake. Pretreatment of RBCMF with phospholipases A and C decreased both Ca⁺⁺ ATPase and Ca⁺⁺ uptake, whereas pretreatment with phospholipase D did not significantly alter either Ca⁺⁺ ATPase or Ca⁺⁺ uptake. Both Ca⁺⁺ ATPase and Ca⁺⁺ uptake had ATP specificity, similar optimum pH's, and optimum incubation temperatures. From these results, it was concluded that Ca⁺⁺ uptake is intimately linked to Ca⁺⁺ ATPase.     

Conformational changes of membrane-bound (Na+−K+)-ATPase as revealed by antibody inhibition

Summary As different structural states of the (Na⁺–K⁺)-ATPase (EC 3.6.1.3) may lead to a changed reactivity to antibodies, the influence of Na⁺, K⁺, Mg⁺⁺, P_i and ATP on the reaction between highly purified (Na⁺–K⁺)-ATPase and antibodies directed against the membrane-bound enzyme was measured. The antigen antibody reaction was registered by measuring the antibody inhibition of (Na⁺–K⁺)-ATPase activity.In themembrane-bound but not in thesolubilized enzyme four different degrees of antibody inhibition were obtained at equilibrium of the antigen antibody reaction if different combinations of Na⁺, K⁺, Mg⁺⁺ and ATP were present during the incubation with the antibodies. Corresponding to the different degrees of inhibition, different rates of enzyme inhibition were measured. (a) The smallest degree of enzyme inhibition was obtained when (i) only Mg⁺⁺, (ii) Mg⁺⁺ and Na⁺ or (iii) Mg⁺⁺ and K⁺ were present during the antigen antibody reaction. (b) The enzyme activity was inhibited more strongly if Na⁺, Mg⁺⁺ and ATP were present together. (c) It was inhibited even more if only (i) Na⁺, (ii) K⁺, (iii) ATP or both (iv) ATP and Na⁺, (v) ATP and K⁺, (vi) ATP and Mg⁺⁺, or if (vii) no ATP and activating ions were present. (d) The highest degree of antibody inhibition was obtained if Mg⁺⁺, ATP and K⁺ were present together.In the presence of Mg⁺⁺ plus ADP and in the presence of Mg⁺⁺ plus the ATP analog adenylyl (--methylene) diphosphonate, Na⁺ and K⁺ did not influence the degree of antibody inhibition as they did in the presence of Mg⁺⁺ plus ATP. It was further found that the degree of antibody inhibition in the presence of Mg⁺⁺, ATP and K⁺ was affected by the sequence in which K⁺ and ATP were added to the enzyme prior to the addition of the antibodies.It is suggested that by antibody inhibition different conformations of the (Na⁺–K⁺)-ATPase could be detected. These conformations may possibly not occur in the solubilized enzyme and therefore do not seem to be necessarily linked to the intermediary steps of the ATP hydrolysis of the enzyme. The structural changes which are induced by Na⁺ and K⁺ in the presence of Mg⁺⁺ plus ATP are proposed to occur during the Na⁺–K⁺ transport.     

Kinetics of iron absorption by excised rice roots

Summary Studies on the rate of iron absorption by excised rice roots from solutions of different concentrations of FeSO₄ showed the presence of two patterns, one in the low (0.005–0.5 mM) and the other in the high (1–30 mM) concentration range. The presence of CaSO₄ or MnSO₄ at 0.5 mM enhanced Fe⁺⁺ absorption in the low concentration range, while CaSO₄ at 10 mM inhibited Fe absorption in the high concentration range in a competitive manner. Fe⁺⁺ absorption at both low and high concentrations was sensitive to metabolic inhibitors. The isotherm for Fe⁺⁺ absorption at O° exhibited an initial absorption shoulder in both low and high concentrations and was suggestive of a latent ion-transport capacity for Fe⁺⁺ in rice roots.     

Induction of contractile activity by rigor complexes in myofibrils

The relation between ATPase rate and substrate concentration was investigated for myofibrils with varying amounts of added HMM. There was a biphasic, 3 to 5-fold increase in ATPase in the absence of Ca⁺⁺. In the absence of added HMM, the peak activity occurred at ≤ 0.1 mM MgATP. With increasing concentrations of HMM, the position and magnitude of the ATPase peak shifted to larger substrate concentrations and higher rates. The cofactor activity of regulated actin in myofibrils is activated to a similar degree by Ca⁺⁺ as by HMM (rigor links). SDS gel electrophoretic patterns of myofibrils mixed with HMM indicated the soluble HMM binds to myofibrils at 0.1 mM MgATP and is dissociated at higher MgATP concentrations. Thus, in well-regulated myofibrils in the absence of Ca⁺⁺ actin cofactor activity can be activated by rigor complexes.     

Requirement of medium ADP for the steady-state hydrolysis of ATP by the proton-translocating Paracoccus denitrificans Fo·F1-ATP synthase

F_o·F₁-ATP synthase in inside-out coupled vesicles derived from Paracoccus denitrificans catalyzes P_i-dependent proton-translocating ATPase reaction if exposed to prior energization that relieves ADP·Mg²⁺-induced inhibition (Zharova, T.V. and Vinogradov, A.D. (2004) J. Biol. Chem.,279, 12319–12324). Here we present evidence that the presence of medium ADP is required for the steady-state energetically self-sustained coupled ATP hydrolysis. The initial rapid ATPase activity is declined to a certain level if the reaction proceeds in the presence of the ADP-consuming, ATP-regenerating system (pyruvate kinase/phosphoenol pyruvate). The rate and extent of the enzyme de-activation are inversely proportional to the steady-state ADP concentration, which is altered by various amounts of pyruvate kinase at constant ATPase level. The half-maximal rate of stationary ATP hydrolysis is reached at an ADP concentration of 8 × 10⁻⁶ M. The kinetic scheme is proposed explaining the requirement of the reaction products (ADP and P_i), the substrates of ATP synthesis, in the medium for proton-translocating ATP hydrolysis by P. denitrificans F_o·F₁-ATP synthase.     

Effects of beta-bungarotoxin on calcium uptake by sarcoplasmic reticulum from rabbit skeletal muscle

A fluorescent chelate probe and a Millipore filtration technique have been used to study the effects of β-bungarotoxin (β-toxin) on passive and active Ca⁺⁺ uptake and ATPase in fragmented sarcoplasmic reticulum (SR) of rabbit skeletal muscle. β-Toxin at 3 × 10^?6 M did not affect ATPase activity. In the absence of ATP, β-Toxin increased the passive uptake of Ca⁺⁺; in the presence of ATP, active Ca⁺⁺ uptake was inhibited. The effect of β-toxin in SR can be detected at concentrations as low as 10^?9 M. The results suggest that β-toxin induces Ca⁺⁺ leakage in SR membranes.     

Über die Spaltung von Adenosintriphosphat in Isolierten Myofibrillen aus Insektenflugmuskeln

Zusammenfassung An isolierten Myofibrillen aus den indirekten Flugmuskeln der Schmeißfliege Phormia regina wurde die Lokalisation der ATPase-Aktivität in der Feinstruktur sowie die Bedingungen, unter denen ihr Nachweis gelingt, untersucht. Die Fällung des freigesetzten Phosphats geschah dabei entweder direkt mit Pb⁺⁺ oder aber zunächst mit Ca⁺⁺ bei nachfolgender Umfällung in Bleiphosphat.Mit beiden Methoden konnte demonstriert werden, daß die Ablagerung des Reaktionsprodukts (und damit die Spaltung von ATP) in zwei verschiedenen Abschnitten der Sarcomere erfolgt: im A-Band und in der Z-Scheibe.Innerhalb des A-Bandes finden sich in der Regel Abschnitte mit unterschiedlicher Niederschlagsdichte. Während die — häufig von der H-Zone zerteilte — Zentralregion mit Präzipitat dicht bedeckt ist, enthalten die Endabschnitte an der Grenze zum I-Band deutlich weniger Partikel. Die Bleiphosphatkristallite sind nicht selten in longitudinalen Reihen angeordnet, die der Position der Primärfilamente entsprechen. In der H-Zone wurden niemals Anzeichen für eine Spaltung von ATP gefunden.Die ATPase-Aktivität der Z-Scheibe läßt sich regelmäßig und unter den verschiedensten Bedingungen demonstrieren. Sie muß deswegen als ein spezifisches Kennzeichen dieser Sarcomerenregion angesehen werden. Es gelang allerdings nicht, die Hydrolyse von ATP in der Z-Scheibe unabhängig von der im A-Band nachzuweisen und umgekehrt.

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Summary The fine structural localization of ATPase activity and the conditions under which it can be demonstrated in myofibrils isolated from indirect flight muscles of the blowfly Phormia regina were studied, using either Pb⁺⁺ or Ca⁺⁺ as precipitating reagents.With both techniques, the deposition of reaction product indicating the hydrolysis of ATP was found in two separate parts of the sarcomere: in the A band and in the Z disc.The A band very often appeared to consist of a central region (frequently divided by the H zone) densely covered with precipitate, while narrow zones bordering the A/I junction contained significantly less particles. Sometimes the precipitated cristallites were found to be arranged in longitudinal rows which corresponded to the position of the thick filaments. Under no conditions was ATPase activity detected in the H zone.The Z disc is established as a specific place of ATP splitting in the sarcomere although it was not possible to demonstrate both myofibrillar ATPase activities independently from each other.

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Dem Andenken an Herrn Prof. Dr. Wolfgang von Buddenbrook gewidmet.

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Wir danken Frau D. Rewicki für ihre kompetente Mithilfe bei der Ausführung der Experimente und der Aufarbeitung des Materials. Der Deutschen Forschungsgemeinschaft sind wir für ihre großzügige Unterstützung der Arbeit zu Dank verpflichtet.     

Divalent cation-activated ATP hydrolysis by mitochondrial ATP'ase — Mechanism for energy depletion in ischemic reperfused myocardium

Recovery of high-energy compounds by ischemic myocardium is believed to be important for its return to normal functioning. While it has been previously shown that oxidative phosphorylation is markedly reduced in mitochondria isolated from ischemic myocardium in the presence of all substrates, alterations in ATPase activity have not been confirmed. This study demonstrates that, although the rate of ATP hydrolysis produced by mitochondria isolated from 2-hr ischemic myocardium does not significantly differ from that produced by non-ischemic mitochondria, the rate produced by 2-hr ischemic, 2 hr reperfused mitochondria is significantly higher. Also, Ca⁺⁺ content was observed to be higher in reperfused than in non-reperfused ischemic mitocheondria. The addition of EDTA, EGTA, or oligomycin to the reperfused ischemic mitochondria resulted in the inhibition of ATPase activity. These results indicate that mitochondrial ATPase in ischemic myocardium is activated by Ca⁺⁺ ions and that ischemic reperfused myocardium may contain mitochondria with uncontrolled ATPase activity such that high energy phosphate supplies are excessively depleted when the cells are reperfused.     

Separate effects of mercurial compounds on the ionophoric and hydrolytic functions of the (Ca+++Mg++)-ATPase of sarcoplasmic reticulum

Summary We have shown that a Ca⁺⁺-ionophore activity is present in the (Ca⁺⁺+Mg⁺⁺)-ATPase of rabbit skeletal muscle sarcoplasmic reticulum (A.E. Shamoo & D.H. MacLennan, 1974.Proc. Nat. Acad. Sci. USA 71:3522). Methylmercuric chloride inhibited the (Ca⁺⁺+Mg⁺⁺)-ATPase and Ca⁺⁺ transport, but had no effect on the activity of the Ca⁺⁺ ionophore. Mercuric chloride inhibited ATPase, transport and ionophore activity. The ATPase and transport functions were more sensitive to methylmercuric chloride than to mercuric chloride. The two functions were inhibited concomitantly by methylmercuric chloride but slightly lower concentrations of mercuric chloride were required to inhibit Ca⁺⁺ transport than were required to inhibit ATPase. Methylmercuric chloride and mercuric chloride probably inhibited ATPase and Ca⁺⁺ transport by blocking essential-SH groups. However, it appears that there are no essential-SH groups in the Ca⁺⁺ ionophore and that mercuric chloride inhibited the Ca⁺⁺ ionophore activity by competition with Ca⁺⁺ for the ionophoric site. Blockage of Ca⁺⁺ transport by mercuric chloride probably occurs both at sites of essential-SH groups and at sites of ionophoric activity. These data suggest the separate identity of the sites of ATP hydrolysis and of Ca⁺⁺ ionophoric activity.     

Active calcium ion uptake by inside-out and right side-out vesicles of red blood cell membranes

The relationship between active extrusion of Ca⁺⁺ from red cell ghosts and active uptake of Ca⁺⁺ by isolated red cell membrane fragments was investigated by studying the Ca⁺⁺ uptake activities of inside-out and right side-out vesicles. Preparations A and B which had mainly inside-out and right side-out vesicles, respectively, were isolated from red cell membranes and were compared with respect to Ca⁺⁺ adenosine triphosphatase (ATPase) and ATP-dependent Ca⁺⁺ uptake activities. Preparation A had nearly eight times more inside-out vesicles and took up eight times more ⁴⁵Ca in the presence of ATP compared to preparation B. Separation of the ⁴⁵Ca-labeled membrane vesicles by density gradient centrifugation showed that the ⁴⁵Ca label was localized to the inside-out vesicle fraction. In addition, the ⁴⁵Ca taken up in the presence of ATP was lost during a subsequent incubation in the absence of ATP. The rate of ⁴⁵Ca loss was not influenced by the presence of EGTA, but was slowed in the presence of La⁺⁸ (0.1 mM) in the efflux medium. The results presented here support the thesis that the active uptake of Ca⁺⁺ by red cell membrane fragments is due to the active transport of Ca⁺⁺ into inside-out vesicles.     

Uncoupling of Occlusion From ATP Hydrolysis Activity in Sarcoplasmic Reticulum (Ca2++Mg2+)-ATPase

The uncoupling of Ca²⁺ transport from ATP hydrolysis in the sarcoplasmic reticulum (Ca²⁺ + Mg²⁺)-ATPase by trypsin digestion was re-investigated by comparing ATPase activity with the ability of the enzyme to occlude Eu³⁺ (a transport parameter) after various tryptic digests. With this method, re-examination of uncoupling by tryptic digest of the ATPase revealed that TD2 cleavage (Arg-198) had no effect on either occlusion or ATPase activity. Digestion past TD2 in the presence of 5 mM Co²⁺ and at 25°C resulted in the loss of about 70% of the ATPase activity, but no loss of occlusion. Digestion past TD2 in the presence of 5 mM Ca²⁺, 3 mM ATP, and at 25°C resulted in a partially uncoupled enzyme complex which retained about 50% of the ATPase activity, but completely lost the ability to occlude Eu³⁺. Digest past TD2 in the presence of 5 mM Ca²⁺ and 3 mM AMP-PNP. (a non-hydrolyzable ATP analog) at 25°C resulted in no loss of occlusion, thus revealing the absolute requirement of ATP during the digest to eliminate occlusion. From these findings we conclude that uncoupling of Ca²⁺ transport from ATPase activity is possible by tryptic digestion of the (Ca²⁺ + Mg²⁺)-ATPase. Interestingly, only after phosphorylation of the enzyme do the susceptible bond(s) which lead to the loss of occlusion become exposed to trypsin.     

Magnesium-dependent conformational changes of membrane proteins are related to the Mg2+ -dependent ATPase activity in cardiac sarcolemma

Magnesium-induced enzymatic and structural changes of membrane-bound proteins in rat heart sarcolemma have been investigated. In the absence of ATP, increasing concentrations of magnesium within the range 0.1–10.0 mM gradually lowered the α-helix content of sarcolemmal proteins. The same magnesium concentrations stepwise activated the Mg²⁺-dependent ATPase in the presence of ATP. Mathematical and graphical analysis of the data yielded a quantitative relationship between magnesium-induced stimulation of the Mg²⁺-dependent ATPase activity and diminution of the α-helix content of membrane proteins in cardiac sarcolemma.     

Oxidative damage to sarcoplasmic reticulum Ca2+-pump induced by Fe2+/H2O2/ascorbate is not mediated by lipid peroxidation or thiol oxidation and leads to protein fragmentation

The major protein in the sarcoplasmic reticulum (SR) membrane is the Ca²⁺ transporting ATPase which carries out active Ca²⁺ pumping at the expense of ATP hydrolysis. The aim of this work was to elucidate the mechanisms by which oxidative stress induced by Fenton's reaction (Fe²⁺ + H₂O₂ HO· + OH^–+ Fe³⁺) alters the function of SR. ATP hydrolysis by both SR vesicles (SRV) and purified ATPase was inhibited in a dose-dependent manner in the presence of 0–1.5 MM H₂O₂ plus 50 M Fe²⁺ and 6 mM ascorbate. Ca²⁺ uptake carried out by the Ca²⁺-ATPase in SRV was also inhibited in parallel. The inhibition of hydrolysis and Ca²⁺ uptake was not prevented by butylhydroxytoluene (BHT) at concentrations which significantly blocked formation of thiobarbituric acid-reactive substances (TBARS), suggesting that inhibition of the ATPase was not due to lipid peroxidation of the SR membrane. In addition, dithiothreitol (DTT) did not prevent inhibition of either ATPase activity or Ca²⁺ uptake, suggesting that inhibition was not related to oxidation of ATPase thiols. The passive efflux of ⁴⁵Ca²⁺ from pre-loaded SR vesicles was greatly increased by oxidative stress and this effect could be only partially prevented (ca 20%) by addition of BHT or DTT. Trifluoperazine (which specifically binds to the Ca²⁺-ATPase, causing conformational changes in the enzyme) fully protected the ATPase activity against oxidative damage. These results suggest that the alterations in function observed upon oxidation of SRV are mainly due to direct effects on the Ca²⁺-ATPase. Electrophoretic analysis of oxidized Ca²⁺-ATPase revealed a decrease in intensity of the silver-stained 110 kDa Ca²⁺-ATPase band and the appearance of low molecular weight peptides (MW < 100 kDa) and high molecular weight protein aggregates. Presence of DTT during oxidation prevented the appearance of protein aggregates and caused a simultaneous increase in the amount of low molecular weight peptides. We propose that impairment of function of the Ca²⁺-pump may be related to aminoacid oxidation and fragmentation of the protein.Abbreviations AcP acetylphosphate - BHT butylhydroxytoluene - DTT dithiothreitol - Hepes 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid - SDS sodium dodecyl sulfate - SDS-PAGE polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate - SR sarcoplasmic reticulum - SRV sarcoplasmic reticulum vesicles - TBA thiobarbituric acid - TBARS thiobarbituric acid-reactive substances - TFP trifluoperazine     

Levels of ornithine decarboxylase activation used as a simple marker of metal induced growth arrest in tissue culture.

Exposure of proliferating cells to specific water solube metal compounds at 0.1 or 1.0 mM concentrations inhibited cell growth and also depressed the induction of ornithine decarboxylase, an enzyme which is tightly coupled to the initiation of cell growth. Salts of Co⁺⁺, Ni⁺⁺, Cu⁺⁺, Cr⁺⁶ and Cd⁺⁺ significantly reduced incorporation of radiolabeled leucine, thymidine or uridine into trichloroacetic acid insoluble material, inhibited the doubling of Chinese hamster ovary cells, and blocked the the induction of ornithine decarboxylase. The addition of similar concentrations of other metals such as Fe⁺⁺, K⁺, Mg⁺⁺, Pb⁺⁺, Ca⁺⁺ or Sn⁺⁺ had no effect on ODC induction and also did not inhibit the other parameters associated with cell proliferation which were measured. These results suggest that ornithine decarboxylase induction can be used as a marker of metal induced growth arrest.