The Ca2+-extruding ATPase of the human platelet creates and responds to cytoplasmic pH changes,consistent with a 2 Ca2+/nH+ exchange mechanism

The Ca²⁺-extruding ATPase pump of the human platelet was studiedin situ by measuring Ca²⁺ extrusion from quin2-overloaded platelets (Johansson, J.S., Haynes, D.H. 1988.J. Membrane Biol. 104:147–163). Cytoplasmic pH (pH_cyt) was measured by BCECF fluorescence in parallel experiments. The pump was studied by raising the cytoplasmic free Ca²⁺ to 2.5 μM and monitoring active Ca²⁺ extrusion into a Ca²⁺-free medium. The pump was shown to perturb pH_cyt, to not respond to changes in membrane potential and to respond to imposed changes in pH_cyt in a manner consistent with the Ca²⁺ pump acting as a 2 Ca²⁺/nH⁺ exchanger. (i) Raising the external pH (pH_ext) from 7.40 to 7.60 lowers the V_max of the pump in basal condition (V_max,1) from 110±18 to 73±12 μM/min (=μmol/liter cell volume/min). (ii) Lowering pH_ext to 7.13 raised V_max,1 to 150±15 μM/min. (iii) In an N-methyl-d-glucamine (NMDG⁺) medium, the pump operation against high [Ca²⁺]_cyt acidifies the cytoplasm by −0.36±0.10 pH units, and the pump becomes self-inhibited. (iv) Use of nigericin to drive pH_cyt down to 6.23 reduces the V_max,1 to 18±11 μM/min. (v) Alkalinization of the cytoplasm by monensin in the presence of Na⁺ raises the V_max,1 (basal state withK _m,1=80 nM) to 136±24 μM/min, but also activates the pump fourfold (V_max,2=280±28 μM/min;K _m,2=502±36 nM). (vi) Transient elevation of pH_cyt by NH₄Cl at high [Ca²⁺]_cyt activates the pump eightfold (V_max,2≥671±350 μM/min). The large activation by alkaline pH_cyt at high [Ca²⁺]_cyt can be explained by Ca²⁺-calmodulin activation of the pump (Valant, P.A., Adjei, P.N., Haynes, D.H. 1992.J. Membrane Biol. 130:63–82) and by increased Ca²⁺ affinity of calmodulin at high pH.     

ATPase activity,IF1 content,and proton conductivity of ESMP from control and ischemic slow and fast heart-rate hearts

Earlier studies by Rouslin and coworkers showed that, during myocardial ischemia in slow heart-rate species which include rabbits and all larger mammals examined including humans, there is an IF₁-mediated inhibition of the mitochondrial ATPase due to an increase in the amount of IF₁, bound to the ATPase (Rouslin, W., and Pullman, M.E.,J. Mol. Cell. Cardiol. 19, 661–668, 1987). Earlier work by Guerrieri and colleagues demonstrated that IF₁ binding to bovine heart ESMP was accompanied by parallel decreases in ATPase activity and in passive proton conduction (Guerrieri, F.,et al., FEBS Lett. 213, 67–72, 1987). In the present study rabbit was used as the slow heart-rate species and rat as the fast heart-rate species. Rat is a fast heart-rate species that contains too little IF₁ to down regulate the ATPase activity present. Mitochondria were prepared from control and ischemic hearts and ESMP were made from aliquots by sonication at pH 8.0 with 2 mM EDTA. Oligomycin-sensitive ATPase activity and IF₁ content were measured in SMP prepared from the control and ischemic mitochondrial samples. After identical incubation procedures, oligomycin-sensitive ATPase activity, oligomycin-sensitive proton conductivity, and IF₁ content were also measured in ESMP samples. The study was undertaken to corroborate further what appear to be fundamental differences in ATPase regulation between slow and fast heart-rate mammalian hearts evident during total myocardial ischemia. Thus, passive proton conductivity was used as an independent measure of these regulatory differences. The results show that, consistent with the low IF₁ content of rat heart cardiac muscle mitochondria, control rat heart ESMP exhibit approximately twice as much passive proton conductivity as control rabbit heart ESMP regardless of the pH of the incubation and assay. Moreover, while total ischemia caused an increase in IF₁ binding and a commensurate decrease in passive proton conductivity in rabbit heart ESMP regardless of pH, neither IF₁ content nor proton conductivity changed significantly in rat heart ESMP as a result of ischemia.This paper is dedicated to the memory of Dr. G. Capozza who died in 1994.     

Proton translocation in cytochromec oxidase: Redox linkage through proximal ligand exchange on cytochromea 3

An analysis of resonance Raman scattering data from CO-bound cytochromec oxidase and from the photodissociated enzyme indicates that histidine may not be coordinated to the iron atom of cytochromea ₃ in the CO-bound form of the enzyme. Instead, the data suggest that either a water molecule or a different amino acid residue occupies the proximal ligand position. From these data, it is postulated that ligand exchange on cytochromea ₃ can occur under physiological conditions. Studies of mutant hemoglobins have demonstrated that tyrosinate binds preferentially to histidine in the ferric forms of the proteins. In cytochromec oxidase tyrosine residues are located near the histidine residues recently implicated in coordination to cytochromea ₃ (Shapleighet al., 1992; Hosleret al., this volume). Expanding on these concepts, we propose a model for proton translocation at the O₂-binding site based on proximal ligand exchange between tyrosine and histidine on cytochromea ₃. The pumping steps take place at the level of the peroxy intermediate and at the level of the ferryl intermediate in the catalytic cycle and are thereby consistent with the recent results of Wilkstrom (1989) who found that proton pumping occurs only at these two steps. It is shown that the model may be readily extended to account for the pumping of two protons at each of the steps.     

Current-voltage characteristics of the proton pump atChara plasmalemma: I. pH dependence

Summary A feature of the current-voltage (I/V) and conductance-voltage (G/V) characteristics is described, which can be attributed to the action of the proton pump at theChara plasmalemma. The study of pH dependence in the range 4.5 to 11.0 and exposure to metabolic inhibitors confirm the pump involvement.A model describing kinetics of H⁺-extruding ATPase (Hansen, U.-P., Gradmann, D., Sanders, D., Slayman, C.L. 1981.J. Membrane Biol. 63:165–190) is fitted to the data yielding theI/V andG/V relationships of theChara proton pump. The results show that the stoichiometry is 1H⁺1ATP.     

Demonstration of the electrogenicity of proton translocation during the phosphorylation step in gastric H+K+-ATPase

Summary Membrane fragments containing the H⁺K^–-ATPase from parietal cells have been adsorbed to a planar lipid membrane. The transport activity of the enzyme was determined by measuring electrical currents via the capacitive coupling between the membrane sheets and the planar lipid film. To initiate the pump currents by the ATPase a light-driven concentration jump of ATP from caged ATP was applied as demonstrated previously for Na⁺K⁺-ATPase (Fendler, K., Grell, E., Haubs, M., Bamberg, E. 1985.EMBO J. 4:3079–3085). Since H⁺K⁺-ATPase is an electroneutrally working enzyme no stationary pump currents were observed in the presence of K⁺. By separation of the H⁺ and K⁺ transport steps of the reaction cycle, however, the electrogenic step of the phosphorylation could be measured. This was achieved in the absence of K⁺ or at low concentrations of K⁺. The observed transient current is ATP dependent which can be assigned to the proton movement during the phosphorylation. From this it was conclueded that the K⁺ transport during dephosphorylation is electrogenic, too, in contrast to the Na⁺K⁺-ATPase where the K⁺ step is electroneutral. The transient current was measured at different ionic conditions and could be blocked by vanadate and by the H⁺K⁺-ATPase specific inhibitor omeprazole. An alternative mechanism for activation of this inhibitor is discussed.     

Mechanism of proton transport by plant plasma membrane proton ATPases

The mechanism of proton translocation by P-type proton ATPases is poorly defined. Asp684 in transmembrane segment M6 of the Arabidopsis thaliana AHA2 plasma membrane P-type proton pump is suggested to act as an essential proton acceptor during proton translocation. Arg655 in transmembrane segment M5 seems to be involved in this proton translocation too, but in contrast to Asp684, is not essential for transport. Asp684 may participate in defining the E₁ proton-binding site, which could possibly exist as a hydronium ion coordination center. A model of proton translocation of AHA2 involving the side chains of amino acids Asp684 and Arg655 is discussed.     

Reaction centers fromRhodopseudomonas sphaeroides in reconstituted phospholipid vesicles. II. Light-induced proton translocation

Unidirectional light-dependent proton translocation was demonstrated in a suspension of reconstituted reaction center (RC) vesicles supplemented with cytochromec and 2,3-dimethoxy-5-methyl-1,4-benzoquinone (UQ₀), a lipid-and water-soluble quinone. Proton translocation was detected only at alkaline pH. The pH dependence can be accounted for by the slow redox reaction between the reduced quinone (UQ₀H₂) and oxidized cytochromec. This conclusion is based on (i) the pH dependence of partial reactions of the reconstituted proton translocation cycle, measured either optically or electrometrically and (ii) titration studies with cytochromec and UQ₀. At 250 and 25 µM UQ₀ and cytochromec, respectively, maximal proton translocation was observed at pH 9.6. This pH optimum can be extended to a more acidic pH by increasing the concentration of the soluble redox mediators in the reconstituted cyclic electron transfer chain. At the alkaline side of the pH optimum, proton translocation appears to be limited by electron transfer from the endogenous primary to the secondary quinone within the RCs. The light intensity limits the reconstituted proton pump at the optimal pH. The results are discussed in the context of a reaction scheme for the cyclic redox reactions and the associated proton translocation events.Abbreviations RC reaction center - UQ₀/UQ₀H₂ oxidized and reduced form of 2,3-dimethoxy-5-methyl-1,4-benzoquinone - D/D⁺ reduced and oxidized form of the primary electron donor of the RCs - CCCP carbonylcyanide-trichloromethoxy phenylhydrazone - UQ_A/UQ _A ^– oxidized and semiquinone form of the primary electron acceptor of the RCs - UQ_B/UQ _B ^– /UQ_BH₂ oxidized, semiquinone, and reduced form of the secondary electron acceptor of the RCs - LDAO lauryldimethylamine-N-oxide During the course of this study K.J.H. was supported by a grant from the Netherlands Organization for the Advancement of Pure Research (Z.W.O.). This research was supported by grants from the National Institutes of Health (EY-02084) and from the Office of Naval Research (ONR-NOOO 14-79-C 0798) to M. Montal.     

Inhibition of gastric acid secretion by a standardized aqueous extract of Cecropia glaziovii Sneth and underlying mechanism

Cecropia glazioui Sneth (Cecropiaceae) is used in folk medicine in tropical and subtropical Latin America as cardiotonic, diuretic, hypotensive, anti-inflammatory and anti-asthmatic. The hypotensive/antihypertensive activity of the plant aqueous extract (AE) and isolated butanolic fraction (BuF) has been confirmed and putatively related to calcium channels blockade in vascular smooth musculature [Lapa, A.J., Lima-Landman, M.T.R., Cysneiros, R.M, Borges, A.C.R., Souccar, C., Barreta, I.P., Lima, T.C.M., 1999. The Brazilian folk medicine program to validate medicinal plants – a topic in new antihypertensive drug research. In: Hostettman, K., Gupta, M.P., Marston, A. (Eds.), Proceedings Volume, IOCD/CYTED Symposium, Panamá City, Panamá, 23–26 February 1997. Chemistry, Biological and Pharmacological Properties of Medicinal Plants from the Americas. Harwood Academic Publishers, Amsterdam, pp. 185–196; Lima-Landman, M.T., Borges, A.C., Cysneiros, R.M., De Lima, T.C., Souccar, C., Lapa, A.J., 2007. Antihypertensive effect of a standardized aqueous extract of Cecropia glaziovii Sneth in rats: an in vivo approach to the hypotensive mechanism. Phytomedicine 14, 314–320]. Bronchodilation and antidepressant-like activities of both AE and BuF have been also shown [Delarcina, S., Lima-Landman, M.T., Souccar, C., Cysneiros, R.M., Tanae, M.M., Lapa, A.J., 2007. Inhibition of histamine-induced bronchospasm in guinea pigs treated with Cecropia glaziovi Sneth and correlation with the in vitro activity in tracheal muscles. Phytomedicine 14, 328–332; Rocha, F.F., Lima-Landman, M.T., Souccar, C., Tanae, M.M., De Lima, T.C., Lapa, A.J., 2007. Antidepressant-like effect of Cecropia glazioui Sneth and its constituents – in vivo and in vitro characterization of the underlying mechanism. Phytomedicine 14, 396–402]. This study reports the antiulcer and antisecretory gastric acid activities of the plant AE, its BuF and isolated compounds with the possible mechanism involved. Both AE and BuF were assayed on gastric acid secretion of pylorus-ligated mice, on acute models of gastric mucosal lesions, and on rabbit gastric H⁺, K⁺-ATPase preparations. Intraduodenal injection of AE or BuF (0.5–2.0 g/kg, i.d) produced a dose-related decrease of the basal gastric acid secretion in 4-h pylorus-ligated mice. At 1.0 g/kg, BuF decreased the volume (28%) and total acidity (33%) of the basal acid secretion, and reversed the histamine (2.5 mg/kg, s.c.)- or bethanecol (1.0 mg/kg, s.c.)-induced acid secretion to basal values, indicating inhibition of the gastric proton pump. Pretreatment of mice with the BuF (0.05–0.5 g/kg, p.o.) protected against gastric mucosal lesions induced by 75% ethanol, indomethacin (30 mg/kg, s.c.) or restraint at 4 °C. BuF also decreased the gastric H⁺, K⁺-ATPase activity in vitro proportionately to the concentration (IC₅₀=58.8 μg/ml). The compounds isolated from BuF, consisting mainly of cathechins, procyanidins and flavonoids [Tanae, M.M., Lima-Landman, M.T.R., De Lima, T.C.M., Souccar, C., Lapa, A.J., 2007. Chemical standardization of the aqueous extract of Cecropia glaziovii Sneth endowed with antihypertensive, bronchodilator, antacid secretion and antidepressant-like activities. Phytomedicine 14, 309–313], inhibited the in vitro gastric H⁺, K⁺-ATPase activity at equieffective concentrations to that of BuF. The results indicate that C. glazioui constituents inhibit the gastric proton pump; this effect may account for the effective antisecretory and antiulcer activities of the standardized plant extract.     

Studies on the tonoplast action potential ofNitella flexilis

Summary The origins of the two peaks of the action potential inNitella flexilis were analyzed by inserting two microelectrodes. one into the vacuole and the other into the cytoplasm. It was unequivocally demonstrated that the rapid first peak was generated at the plasmalemma and the slow second peak at the tonoplast. MnCl₂ applied in the external medium abolished the second, tonoplast, peak but not the first, plasmalemma, peak, MnCl₂ also inhibited the cessation of the cytoplasmic streaming accompanying the action potential. CaCl₂ added in MnCl₂-containing medium recovered generation of the tonoplast action potential and the streaming cessation. Since it has been established that the cessation of cytoplasmic streaming on membrane excitation is caused by an increase in cytoplasmic free Ca^2– (Williamson, R.E., Ashley, C.C., 1982.Nature (London) 296:647–651: Tominaga, Y., Shimmen, T., Tazawa, M., 1983,Protoplasma 116:75–77), it is suggested that the tonoplast action potential is also induced by an increase in cytoplasmic Ca²⁺ resulting from the plasmalemma excitation. When vacuolar Cl was replaced with SO ₄ ² by vacuolar perfusion, the polarity of the second, slow peak was reversed from vacuolar positive to vacuolar negative with respect to the cytoplasm, supporting the previous report that the tonoplast action potential is caused by increase in Cl permeability (Kikuyama, M., Tazawa, M., 1976.J. Membrane Biol.29:95–110).     

Studies on the perfused plasmalemma ofChara corallina: I. Current-voltage curves: ATP and potassium dependence

Summary The electrical properties of theChara cell membrane have been studied using a perfusion method based on that of Williamson, R.E. 1975.J. Cell Sci. 17655. The vacuole, tonoplast, and inner cytoplasm are removed by a brief rapid perfusion. Electrical properties of the plasmalemma indicate that it remains intact after this perfusion.The membrane potential difference after perfusion and with no ATP was close to the potassium equilibrium potential; the current-voltage characteristic had a slope that was time- and voltage-dependent, indicating that the steady-state potassium conductance increased with depolarization. At –125 mV the membrane conductance of the plasmalemma depended on [K⁺]₀. This dependence was inhibited by perfusing with 2.0mm ATP or by clamping at a more negative membrane potential. The addition of ATP to the perfusion medium of unclamped cells caused a hyperpolarization ofca. 50 mV, presumably by activating the proton pump. In clamped cells, perfusion with ATP caused currents ofca. 20 mA m^–2, whose magnitude depended on pH₀. ATP induced membrane conductance changes which were variable. 2.0mm ADP inhibited the proton pump. The intersection points of current-voltage characteristics can set limits on the stalling potential; the resulting stoichiometry of the proton pump appears to be 1.5–2.0 H⁺ per ATP.     

The voltage-gated Ca2+ channel is the Ca2+ sensor of fast neurotransmitter release

Previously it demonstrated that in the absence of Ca²⁺ entry, evoked secretion occurs neither by membrane depolarization, induction of [Ca²⁺] _i rise, nor by both combined (Ashery, U., Weiss, C., Sela, D., Spira, M. E., and Atlas, D. (1993). Receptors Channels 1:217–220.). These studies designate Ca²⁺ entry as opposed to [Ca²⁺] _i rise, essential for exocytosis. It led us to propose that the channel acts as the Ca²⁺ sensor and modulates secretion through a physical and functional contact with the synaptic proteins. This view was supported by protein–protein interactions reconstituted in the Xenopus oocytes expression system and release experiments in pancreatic cells (Barg, S., Ma, X., Elliasson, L., Galvanovskis, J., Gopel, S. O., Obermuller, S., Platzer, J., Renstrom, E., Trus, M., Atlas, D., Streissnig, G., and Rorsman, P. (2001). Biophys. J.; Wiser, O., Bennett, M. K., and Atlas, D. (1996). EMBO J. 15:4100–4110; Wiser, O., Trus, M., Hernandez, A., Renström, E., Barg, S., Rorsman, P., and Atlas, D. (1999). Proc. Natl. Acad. Sci. U.S.A. 96:248–253). The kinetics of Ca_v1.2 (Lc-type) and Ca_v2.2 (N-type) Ca²⁺ channels were modified in oocytes injected with cRNA encoding syntaxin 1A and SNAP-25. Conserved cysteines (Cys271, Cys272) within the syntaxin 1A transmembrane domain are essential. Synaptotagmin I, a vesicle-associated protein, accelerated the activation kinetics indicating Ca_v2.2 coupling to the vesicle. The unique modifications of Ca_v1.2 and Ca_v2.2 kinetics by syntaxin 1A, SNAP-25, and synaptotagmin combined implied excitosome formation, a primed fusion complex of the channel with synaptic proteins. The Ca_v1.2 cytosolic domain Lc_753–893, acted as a dominant negative modulator, competitively inhibiting insulin release of channel-associated vesicles (CAV), the readily releasable pool of vesicles (RRP) in islet cells. A molecular mechanism is offered to explain fast secretion of vesicles tethered to SNAREs-associated Ca²⁺ channel. The tight arrangement facilitates the propagation of conformational changes induced during depolarization and Ca²⁺-binding at the channel, to the SNAREs to trigger secretion. The results imply a rapid Ca²⁺-dependent CAV (RRP) release, initiated by the binding of Ca²⁺ to the channel, upstream to intracellular Ca²⁺ sensor thus establishing the Ca²⁺ channel as the Ca²⁺ sensor of neurotransmitter release.     

Silencing of genes involved in Anaplasma marginale-tick interactions affects the pathogen developmental cycle in Dermacentor variabilis

Background  

The cattle pathogen, Anaplasma marginale, undergoes a developmental cycle in ticks that begins in gut cells. Transmission to cattle occurs from salivary glands during a second tick feeding. At each site of development two forms of A. marginale (reticulated and dense) occur within a parasitophorous vacuole in the host cell cytoplasm. However, the role of tick genes in pathogen development is unknown. Four genes, found in previous studies to be differentially expressed in Dermacentor variabilis ticks in response to infection with A. marginale, were silenced by RNA interference (RNAi) to determine the effect of silencing on the A. marginale developmental cycle. These four genes encoded for putative glutathione S-transferase (GST), salivary selenoprotein M (SelM), H+ transporting lysosomal vacuolar proton pump (vATPase) and subolesin.     

1H, 15N and 13C resonance assignments and secondary structure of group II phospholipase A2 from Agkistrodon piscivorus piscivorus: Presence of an amino-terminal helix in solution

Summary ¹H, ¹⁵N and ¹³C resonance assignments are presented for the group II phospholipase A2 (PLA2) from Agkistrodon piscivorus piscivorus. The secondary structure of the enzyme has been inferred from an analysis of coupling constants, interproton distances, chemical shifts, and kinetics of amide exchange. Overall, the secondary structure of this PLA2 is similar to the crystal structure of the homologous group II human nonpancreatic secretory phospholipase [Scott, D.L., White, S.P., Browning, J.L., Rosa, J.J., Gelb, M.H. and Sigler, P.B. (1991) Science, 254, 1007–1010]. In the group I enzyme from porcine pancreas, the amino-terminal helix becomes fully ordered in the ternary complex of enzyme, lipid micelles and inhibitor. The formation of this helix is thought to be important for the increase in activity of phospholipases on aggregated substrates [Van den Berg, B., Tessari, M., Boelens, R., Dijkman, R., De Haas, G.H., Kaptein, R. and Verheij, H.M. (1995) Nature Struct. Biol., 2, 402–406]. However, the group II enzyme from Agkistrodon piscivorus piscivorus possesses a defined and well-positioned aminoterminal helix in the absence of substrate. Therefore, there is a clear difference between the conformations of group I and group II enzymes in solution. These conformational differences suggest that formation of the amino-terminal helix is a necessary, but not sufficient, step in interfacial activation of phospholipases.Abbreviations PLA2 phospholipase A2 - App-D49 phospholipase from Agkistrodon piscivorus piscivorus - NOE nuclear Overhauser effect     

‘Random coil’ 1H chemical shifts obtained as a function of temperature and trifluoroethanol concentration for the peptide series GGXGG

Summary Proton chemical shifts of a series of disordered linear peptides (H-Gly-Gly-X-Gly-Gly-OH, with X being one of the 20 naturally occurring amino acids) have been obtained using 1D and 2D ¹H NMR at pH 5.0 as a function of temperature and solvent composition. The use of 2D methods has allowed some ambiguities in side-chain assignments in previous studies to be resolved. An additional benefit of the temperature data is that they can be used to obtain ‘random coil’ amide proton chemical shifts at any temperature between 278 and 318 K by interpolation. Changes of chemical shift as a function of trifluoroethanol concentration have also been determined at a variety of temperatures for a subset of peptides. Significant changes are found in backbone and side-chain amide proton chemical shifts in these ‘random coil’ peptides with increasing amounts of trifluoroethanol, suggesting that caution is required when interpreting chemical shift changes as a measure of helix formation in peptides in the presence of this solvent. Comparison of the proton chemical shifts obtained here for H-Gly-Gly-X-Gly-Gly-OH with those for H-Gly-Gly-X-Ala-OH [Bundi, A. and Wüthrich, K. (1979) Biopolymers, 18, 285–297] and for Ac-Gly-Gly-X-Ala-Gly-Gly-NH₂ [Wishart, D.S., Bigam, C.G., Holm, A., Hodges, R.S. and Sykes, B.D. (1995) J. Biomol. NMR, 5, 67–81] generally shows good agreement for CH protons, but reveals significant variability for NH protons. Amide proton chemical shifts appear to be highly sensitive to local sequence variations and probably also to solution conditions. Caution must therefore be exercised in any structural interpretation based on amide proton chemical shifts.     

Synthesis of 2′,3′-Dideoxy-2′-fluoro-3′-thioarabinothymidine and Its 3′-Phosphoramidite Derivative

Abstract

An efficient method for the synthesis of 5′-O-monomethoxytrityl-2′,3′-dideoxy-2′-fluoro-3′-thioarabinothymidine [^5′-MMTaraF-T_3′SH, (5)] and its 3′-phosphoramidite derivative (6) suitable for automated incorporation into oligonucleotides, is demonstrated. A key step in the synthesis involves reaction of 5′-O-MMT-2,3′-O-anhydrothymidine (4) (Eleuteri, A.; Reese, C.B.; Song, Q., J. Chem. Soc. Perkin Trans. 1 1996, 2237 pp.) with sodium thioacetate to give ^5′-MMTaraF-T_3′SAc (5) (Elzagheid, M.I.; Mattila, K.; Oivanen, M.; Jones, B.C.N.M.; Cosstick, Lönnberg, H. Eur. J. Org. Chem. 2000, 1987–1991). This nucleoside was then converted to its corresponding phosphoramidite derivative, 6, as described previously ((a) Sun, S.; Yoshida, A.; Piccirilli, J.A. RNA, 1997, 3, 1352–1363; (b) Matulic-Adamic, J.; Beigelman, L. Helvetica Chemica Acta 1999, 82, 2141–2150; (c) Fettes, K.J.; O’Neil, I.; Roberts, S.M.; Cosstick, R. Nucleosides, Nucleotides and Nucl. Acids 2001, 20, 1351–1354).     

Evaluation of the antifungal and plasma membrane H+-ATPase inhibitory action of ebselen and two ebselen analogs in S. cerevisiae cultures

The plasma membrane H⁺-ATPase pump (Pma1p) has been proposed as a viable target for antifungal drugs since this high capacity proton pump plays a critical role in the intracellular regulation of pH and in nutrient uptake of yeast and other fungi. In recent years, this and other laboratories have verified that the antifungal activity of 2-phenylbenzisoselenazol-3(2H)-one, an organoselenium compound commonly referred to as ebselen (1), stems, at least in part, from its inhibitory action on the fungal Pma1p. In the present study, the antifungal efficacy of 2-(3-pyridinyl)-benzisoselenazol-3(2H)-one (2) and 2-phenylbenzisoselenazol-3(2H)-one 1-oxide (3), two ebselen analogs, was evaluated using a strain of S. cerevisiae and compared against that of 1. In addition, the study also examined the inhibitory potential of these three compounds toward the Pma1p of S. cerevisiae. Based on mean IC₅₀ values, the antifungal potency was found to decrease in the order 3?>?1?>?2. However, in terms of inhibitory action on Pma1p, the potency decreased in the order 1?>?3?>?2. The magnitude of these activities appears to be correlated with the corresponding log P values, with compound 2 being the most hydrophilic and the least active of the three.     

The pentaammineruthenium(III)histidine complex in ribonuclease A: application to the assignment of histidine proton resonances

The assignment of two histidine proton resonances in the proton NMR spectrum of ribonuclease A has been made by forming a paramagnetic complex between pentaammineruthenium(III) and the N-3 nitrogen of a single histidine residue. Reaction of chloropentaammineruthenium(III)dichloride with ribonuclease A in 0.1 m Tris-HCl, pH 7.0, 25°C yields a variety of products in which various histidine residues have been labeled. Cation-exchange chromatography affords the isolation of a specific derivative, labeled at a single histidine residue, that retains 66% of the activity toward the hydrolysis of 2′,3′-cyclic CMP. The site of labeling was determined by peptide mapping to be histidine 105. The binding of ruthenium results in the disappearance of both a histidine C-2 and a C-4 proton resonance from the downfield region of the proton NMR spectrum, as expected from model compound studies. The assignment of these two resonances to histidine 105 is in agreement with a previous assignment (J. L. Markley, 1975, Biochemistry, 14, 3546–3554), thereby demonstrating the potential utility of this ruthenium reagent in the assignment of histidine resonances in the proton NMR spectra of other proteins.     

Identification of phospholipase D from cabbage as N-terminally acetylated PLD2

Recently, the genes of two isoenzymes of phospholipase D from white cabbage (PLD1 and PLD2) with molecular masses of 91.7 and 91.9 kDa, respectively, have been sequenced and expressed in Escherichia coli [Schäffner, I., Rücknagel, K.-P., Mansfeld, J., and Ulbrich-Hofmann, R. (2002). Eur. J. Lipid Sci. Technol. 104: 79–87]. Both enzymes are highly homologous (91% identity) and behave very similarly. Phospholipase D purified from white cabbage leaves (PLD_cab) is compared with the two recombinant enzymes in sodium dodecylsulfate and native polyacrylamide gel electrophoresis, isoelectric focusing, N-terminal sequencing, and mass spectrometry after tryptic digestion. As a result, PLD_cab clearly can be assigned to PLD2. In contrast to recombinant PLD2, however, PLD_cab is N-terminally acetylated.     

Conformational analysis of thyrotropin releasing factor by proton magnetic resonance spectroscopy

The proton magnetic resonance spectrum of thyrotropin releasing factor (TRF) in solution in deuterium oxide and deuterated dimethylsulfoxide (DMSO–d₆) has been analyzed. Two forms differing in cis–trans isomerism about the His-Pro peptide bond are observed. From the temperature dependence of chemical shift of the amide protons, it is concluded that TRF in DMSO–d₆ does not contain intramolecular hydrogen bonds. Measurement of NH? C_αH coupling constant provides an estimate of the histidine dihedral angle ?. Structural information about the histidine side-chain is deduced from C_αH? C_βH coupling constants and from the nonequivalence of the two prolyl δ-protons. In DMSO–d₆, there is evidence for a tautomeric equilibrium corresponding to an exchange of imidazole proton between the two nitrogen atoms N-δ and N-ε. In water, the N-εH tautomer is found to be the predominant tautomeric form of the imidazole ring. These results in combination with energy calculation, vibrational analysis, and carbon nmr studies allow the determination of the conformationof TRF.     

Cytochrome c Oxidase as a Proton-Pumping Peroxidase: Reaction Cycle and Electrogenic Mechanism

Cytochrome oxidase (COX) is considered to integrate in a single enzyme two consecutive mechanistically different redox activities--oxidase and peroxidase--that can be catalyzed elesewhere by separate hemoproteins. From the viewpoint of energy transduction, the enzyme is essentially a proton pumping peroxidase with a built-in auxiliary eu-oxidase module that activates oxygen and prepares in situ H₂O₂, a thermodynamically efficient but potentially hazardous electron acceptor for the proton pumping peroxidase. The eu-oxidase and peroxidase phases of the catalytic cycle may be performed by different structural states of COX. Resolution of the proton pumping peroxidase activity of COX and identification of individual charge translocation steps inherent in this reaction are discussed, as well as the specific role of the two input proton channels in proton translocation.