Kinetics of conformational changes in tRNAPhe (yeast) as studied by the fluorescence of the y-base and of formycin substituted for the 3'-terminal adenine

The kinetics of the melting transitions of tRNA^phe (yeast) were followed by the fluorescence of the Y-base and of formycin substituted for the 3'-terminal adenine. As judged from differential UV absorbance melting cutves the formycin label had virtually no influence on the conformation of the tRNA. A temperature jump apparatus was modified to allow the simultaneous observation of transmission and fluorescence intensities by two independent optical channels. The design of a temperature jump cell with an all quartz center piece is given. The cell is resistant to temperatures up to 90°C; it provides high optical sensitivity, low stray light intensity and the possibility of measuring fluorescence polarization. The T-jump experiments allowed to discriminate between fast unspecific fluorescence quenching (τ <5 μsec) and slow co-operative conformational changes. In the central part of the temperature range of UV-melung (midpoint temperature 30°C in 0.01 M Na⁺ and 39°C in 0.03 M Na⁺, pH 6.8) two resolvable relaxation processes were observed. The coirssponding relaxation times were 20 msec and 800 msec at 30°C in 0.01 M Na⁺, and 4 msec and 120 msec at 39°C in 0.03 M Na⁺. The Y-base fluorescence shows both of the relaxation effects, which almost cancel in equilibrium fluorescence melting, because their amplitudes have opposite signs. From this finding the existence of some residual tertiary structure is inferred which persists after the unfolding of the main part of tertiary structure durirg early melting (midpoint temperature 24°C in 0.03 M Na⁺). In the fluorescence sigXXX of the formycin also the two relaxation effects appear. Both of them are connected with a decrease of the fluorescence intensity. From the results a coupled opening of the anticodon and acceptor branches is concluded.Enzymes: phenylalanyl-tRNA synthetase, PRS (EC 6.1.1.-20); ATP (CTP) tRNA nucleotidyl transferase, NT (EC 2.7.7.-20); alkaline phosphatase (EC 3-1-3.1).     

Effect of extracellular Ca2+, K+ and OH− on erythrocyte membrane potential as monitored by the fluorescent probe 3,3′-dipropylthiodicarbocyanine

Changes in fluorescence intensity of thiodicarbocyanine, DiS-C₃(5), were correlated with direct microelectrode potential measurements in red blood cells from Amphiuma means and applied qualitatively to evaluate the effects of extracellular Ca²⁺, K⁺ and pH on the membrane potential of human red cells. Increasing extracellular [Ca²⁺] from 1.8 to 15 mM causes a K⁺-dependent hyperpolarization and decrease in fluorescence intensity in Amphiuma red cells. Both the hyperpolarization and fluorescence change disappear when the temperature is raised from 17 to 37°C. No change in fluorescence intensity is observed in human red cells with comparable increase in extracellular Ca²⁺ in the temperature range 5–37°C. Increasing the extracellular pH, however, causes human red cells to respond to an increase in extracellular Ca²⁺ with a significant but temporary loss in fluorescence intensity. This effect is blocked by EGTA, quinine or by increasing extracellular [K⁺], indicating that at elevated extracellular pH, human erythrocytes respond to an increase in extracellular Ca²⁺ with an opening of K⁺ channels and associated hyperpolarization of the plasma membrane.     

Passive potassium ion permeability of Halobacterium halobium cell envelope membranes

Cell envelope vesicles, prepared from Halobacterium halobium, were loaded with 3 M KCl suspended in 3 M NaCl, and the loss of K⁺ was followed at various temperatures. The Arrhenius plot of the K⁺-efflux rates shows a break at 30°C, with higher energy of activation above the break. This temperature dependence is consistent with earlier studies of chain motions in liposomes prepared from isolated lipids. The efflux of K⁺ is more rapid with increasing pH between pH 5 and 7. Since these vesicles do not respire under the experimental conditions it was expected that the K⁺-efflux data would be related to the passive permeability of the membranes to K⁺. The apparent K⁺ permeability at 30°C is 1–2· 10^?10 cm·^?1. This value corresponds to a 5-h half-life for retained K⁺ in the envelope vesicles and to a probably much longer half-life in whole cells. The previously observed ability of Halobacterium to retain K⁺ in the absence of metabolism can thus be explained solely by the permeability characteristics of the membranes.     

Effect of temperature,salts, pH and other factors on the development of peritrichous flagella in Vibrio alginolyticus

The development of peritrichous flagella and, consequently, swarming of Vibrio alginolyticus depend on a complex relationship between temperature, salt concentrations and pH. At temperatures above 28°C V. alginolyticus did not develop peritrichous flagella unless certain minimal concentrations of NaCl are present: the higher the temperature, the higher the NaCl concentrations required for peritrichous flagella synthesis. This requirement for NaCl at high temperatures is much more pronounced at pH 9 than at pH 6. High temperatures and low concentrations of NaCl also inhibited swarming of cells already armed with peritrichous flagella. Other cations, such as Li⁺, K⁺ and Mg²⁺, replaced NaCl only at temperatures below 28°C.     

Effect of root environment on proton efflux in wheat roots

Proton net efflux of wheat (Triticum aestivum L.) roots growing in sand culture or hydroponics was determined by measuring the pH values of the solution surrounding the roots by pH microelectrodes, by base titration and by color changes of a pH indicator in solid nutrient media. The proton net efflux was dependent on light, aeration, and source of nitrogen (NH ₄ ⁺ , NO ₃ ^? ). Ammonium ions caused the highest proton efflux, whereas nitrate ions decreased the proton efflux. Iron deficiency had no significant effect on proton efflux. Replacement of ammonium by nitrate inhibited proton efflux, whereas the reverse enhanced proton extrusion. A lag period between changes in plant environment and proton efflux was observed. The proton net efflux occurred at the basal portion of the roots but not in the root tips or at the elongation zone. Under optimal conditions, proton efflux capacity reached a maximum value of 5.7 μmole H⁺ g^?1 fresh weight h^?1 with an average (between different measurements) of 3.4 μmole H⁺ g^?1 fresh wth^?1 whereas the pH value decreased to 3.2–3.7 and reached a minimal value of 2.9. Inhibition of ATPase activity by orthovanadate inhibited proton efflux. The results indicate that proton efflux in wheat roots is ammonium ion and light dependent and probably governed by ATPase activity.     

The lipid integrity of membranes of guinea pig alveolar macrophages studied by nanosecond fluorescence decay of 1,6-diphenyl-1,3,5-hexatriene: The effect of stimulation by concanavalin A and formyl peptides1

Time-resolved fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene was used to monitor physical changes in the membranes of guinea pig alveolar macrophages following stimulation by N-formyl peptides (either N-formylmethionylphenylalanine (FMP) or N-formyl methionyl leucylphenylalanine (FMLP)) and concanavalin A. The anisotropy of diphenylhexatriene in macrophages showed a dependence on stimulation both in the rate of decay and in the value of anisotropy at infinite time. Subtle differences were observed between the effect of concanavalin A and FMLP on the membrane lipid fluidity as detected by fluorescence anisotropy. Concanavalin A stimulation of macrophages decreased the value of the anisotropy at infinite times in the range of 0–20 °C and increased the value at 25–40 °C; and at all temperatures it decreased the rate of decay of anisotropy. At temperatures below 25 °C, the response to FMLP was similar to concanavalin A, but above 25 °C, FMLP only slightly modified the anisotropy decay profile. Another physical parameter, calcium permeability, was examined because Ca⁺² fluxes are dependent upon membrane properties. The temperature-dependent profiles of concanavalin A and FMP-stimulated ⁴⁵Ca⁺² efflux from alveolar macrophages were similar. The rate and extent of ⁴⁵Ca⁺² efflux increased from 4 to 22 °C, with no further increases observed up to 37 °C. This pattern correlated well with observed changes in membrane fluidity.     

Further investigation on the lateral and transversal proton currents at the thylakoid membrane level by hydrogen-deuterium exchange

Energetically-coupled processes (electron flow, proton uptake and correlated pH gradient) were investigated on envelope-free chloroplasts of lettuce suspended in ¹H₂O or ²H₂O media. Study of the light-intensity and temperature dependencies of these phenomena led to the following observations: 1. At neutral pH, ²H₂O diminishes the transmembrane H⁺ gradient in strong light (chain Photosystem II + Photosystem I) but not in low light; the total H⁺ uptake is increased at all light intensities: the buffering capacity of the inner compartment is increased in heavy water, possibly through enhancement of interactions between membranous titrable groups and the aqueous phase. 2. ²H₂O does not affect the photochemical events of the redox chain, whatever the electron pathway (PSII, PSI or PSII + PSI): only thermal steps are inhibited. The diminution of the apparent quantum yield, sometimes observed, may be ascribed to the dual site of action of the artificial redox carrier (ferricyanide) then used. 3. ²H₂O does not modify the activation energy of the limiting step of the electron flow (PSII + PSI) in uncoupled (44 vs. 47 kJ · mol^?1) or — but less clearly — in coupled, i.e., ‘basal’, state (55 vs. 59 kJ · mol^?1). ²H₂O does not either change the temperature of the phase transition of the membrane (17°C) for the uncoupled flow. However, a low-temperature transition, observed only for the coupled chain, is slightly increased by ²H₂O; this thermal transition is attributed to the freezing of some bound water near the plastoquinone pool. 4. Δp²H is smaller than Δp¹H at all temperatures (PSII + PSI chain). ΔpH is quasi-constant from 0°C to 10°C, then decreases when temperature rises. ²H₂O does not change the activation energy of the dark passive H⁺ efflux, which is almost twice that of the coupled electron flow. The phase transition at low temperature suggests that the proton efflux occurs via two parallel pathways, one temperature-dependent and the other temperature-independent. Except for the increase of the internal buffering capacity, the effects of ²H₂O on the membrane conformation seem limited, as shown by the unchanged activation energies of the electron flow and of the H⁺ leakage. The null activation energy observed at low temperature emphasizes the role of the bound water in these processes; however, the different effects of ²H₂O on the transition temperatures indicate that this bound water has different properties when associated with the translocation sites or with the H⁺ leakage ones. This ‘microcompartmentation’ of the membranes is consistent with the concept of lateral pH heterogeneity we have previously suggested (de Kouchkovsky, Y., and Haraux, F. (1981) Biochem. Biophys. Res. Commun. 99, 205–212). The theoretical computations and the experimental results suggest that in the steady state, the internal pH would be several tenths of a ‘unit’ lower near the plastoquinones than near the H⁺ efflux sites (coupling factors); this difference would be increased when ²H₊ replaces ¹H⁺, owing to the lower mobility of the deuteron. It is concluded that local, and not average, pH (and ΔpH) should be considered for the understanding of the energy transduction processes.     

The kinetics of light-induced changes of C-550, cytochrome b559 and fluorescence yield in chloroplasts at low temperature

The kinetics of the photoreduction of C-550, the photooxidation of cytochrome b₅₅₉ and the fluorescence yield changes during irradiation of chloroplasts at ?196 °C were measured and compared. The photoreduction of C-550 proceeded more rapidly than the photooxidation of cytochrome b₅₅₉ and the fluorescence yield increase followed the cytochrome b₅₅₉ oxidation. These results suggest that fluorescence yield under these conditions indicates the dark reduction of the primary electron donor to Photosystem II, P680⁺, by cytochrome b₅₅₉ rather than the photoreduction of the primary electron acceptor.The photoreduction of C-550 showed little if any temperature dependence over the range of ?196 to ?100 °C. The amount of cytochrome b₅₅₉ photooxidized was sensitive to temperature decreasing from the maximal change at temperatures between ?196 to ?160 °C to no change at ?100 °C. To the extent that the reaction occurred at temperatures between ?160 and ?100 °C the rate was largely independent of temperature. The rate of the fluorescence increase was dependent on temperature over this range being 3–4 times more rapid at ?100 than at ?160 °C. At ?100 °C the light-induced fluorescence increase and the photoreduction of C-550 show similar kinetics. The temperature dependence of the fluorescence induction curve is attributed to the temperature dependence of the dark reduction of P680⁺.The intensity dependence of the photoreduction of C-550 and of the photooxidation of cytochrome b₅₅₉ are linear at low intensities (below 200 μW/cm²) but fall off at higher intensities. The failure of reciprocity in the photoreduction of C-550 at the higher intensities is not explained by the simple model proposed for the Photosystem II reaction centers.     

Alterations in phospholipid-dependent (Na++K+)-ATPase activity due to lipid fluidity: Effects of cholesterol and Mg2+

The (Na⁺+K⁺)-activated, Mg²⁺-dependent ATPase from rabbit kidney outer medulla was prepared in a partially inactivated, soluble from depleted of endogenous phospholipids, using deoxycholate. This preparation was reactivated 10 to 50-fold by sonicated liposomes of phosphatidylserine, but not by non-sonicated phosphatidylserine liposomes or sonicated phosphatidylcholine liposomes. The reconstituted enzyme resembled native membrane preparations of (Na⁺+K⁺)-ATPase in its pH optimum being around 7.0 showing optimal activity at Mg²⁺: ATP mol ratios of approximately 1 and a K_m value for ATP of 0.4 mM.Arrhenius plots of this reactivated activity at a constant pH of 7.0 and an Mg²⁺: ATP mol ratio of 1:1 showed a discontinuity (sharp change of slope) at 17 °C, With activation energy (E_a) values of 13–15 kcal/mol above this temperature and 30–35 kcal below it. A further discontinuity was also found at 8.0 °C and the E_a below this was very high (> 100 kcal/mol).Incresed Mg²⁺ concentrations at Mg²⁺: ATP ratios in excess of 1:1 inhibited the (Na⁺+K⁺)-ATPase activity and also abolished the discontinuities in the Arrhenius plots.The addition of cholesterol to phosphatidylserine at a 1:1 mol ratio partially inhibited (Na⁺+K⁺)-ATPase reactivation. Arrhenius plots under these conditions showed a single discontinuity at 20°C and E_a values of 22 and 68kcal/mol above and below this temperature respectively. The ouabain-insensitive Mg²⁺-ATPase normally showed a linear Arrhenius plot with an E_a of 8 kcal/mol. The cholesterol-phosphatidylserine mixed liposomes stimulated the Mg²⁺-ATPase activity, which now also showed a discontinuity at 20 °C with, however, an increased value of 14 kcal/mol above this temperature and 6 kcal/mol below. Kinetic studies showed that cholesterol had no significant effect on the K_m for ATP.Since both of cholesterol and Mg²⁺ are know to alter the effects of temperature on the fluidity of phospholipids the above result are discussed in this context.     

Acclimatization of K+ uptake to changes in root temperature: experiments with oilseed rape and barley in flowing solution culture

Abstract Changes in the net uptake rate of K⁺ and in the average tissue concentration of K⁺ were measured over 14 d in response to changes in root temperature with oilseed rape (Brassica napus L. cv. Bien venu) and barley (Hordeum vulgare L. cv. Atem). Plants were grown in flowing nutrient solutions containing 2.5 mmol m^?3 K⁺ and were acclimatized over 49 d (rape) or 28 d (barley) to low root temperature (5°C) prior to steady–state treatments at root temperatures between 3 °C and 25 °C, with common air temperature. Uptake of K⁺ was monitored continuously over 14 d and nitrogen was supplied as NH₄⁺+ NO^?₃ or NH⁺₄ or NO^?₃. Unit absorption rates of K⁺ increased with time and with root temperature up to Day 4 or 5 following the change in root temperature. Thereafter they usually approached steady-state, with Q₁₀? 2.0 between 7 °C and 17°C, although rates became similar between 7 °C and 13°C. Uptake of K⁺ by rape plants was invariably greater under NO^?₃ nutrition compared with NH⁺₄. The percentage K⁺ in the plant dry matter increased with temperature from 2% at 3 °C to 4% at 25 °C in rape, but there was less effect of temperature on the average concentrations of K⁺ in the plant fresh weight or plant water content. Concentrations of K⁺ in the leaf water fraction of rape plants decreased with increasing root temperature, but in barley they increased with increasing root temperature. Concentrations of K⁺ in the root water fraction were relatively stable with respect to root temperature. The results are discussed in terms of compensatory changes in K⁺ uptake following a change in root temperature and the relationships between growth, shoot: root ratio and K⁺ composition of the plant.     

NaCl Induces a Na/H Antiport in Tonoplast Vesicles from Barley Roots

Evidence was found for a Na⁺/H⁺ antiport in tonoplast vesicles isolated from barley (Hordeum vulgare L. cv California Mariout 72) roots. The activity of the antiport was observed only in membranes from roots that were grown in NaCl. Measurements of acridine orange fluorescence were used to estimate relative proton influx and efflux from the vesicles. Addition of MgATP to vesicles from a tonoplast-enriched fraction caused the formation of a pH gradient, interior acid, across the vesicle membranes. EDTA was added to inhibit the ATPase, by chelating Mg²⁺, and the pH gradient gradually dissipated. When 50 millimolar K⁺ or Na⁺ was added along with the EDTA to vesicles from control roots, the salts caused a slight increase in the rate of dissipation of the pH gradient, as did the addition of 50 millimolar K⁺ to vesicles from salt-grown roots. However, when 50 millimolar Na⁺ was added to vesicles from salt-grown roots it caused a 7-fold increase in the proton efflux. Inclusion of 20 millimolar K⁺ and 1 micromolar valinomycin in the assay buffer did not affect this rapid Na⁺/H⁺ exchange. The Na⁺/H⁺ exchange rate for vesicles from salt-grown roots showed saturation kinetics with respect to Na⁺ concentration, with an apparent K_m for Na⁺ of 9 millimolar. The rate of Na⁺/H⁺ exchange with 10 millimolar Na⁺ was inhibited 97% by 0.1 millimolar dodecyltriethylammonium.     

Production,characteristics and applications of phytase from a rhizosphere isolated <Emphasis Type="Italic">Enterobacter</Emphasis> sp. ACSS

Optimization of process parameters for phytase production by Enterobacter sp. ACSS led to a 4.6-fold improvement in submerged fermentation, which was enhanced further in fed-batch fermentation. The purified 62 kDa monomeric phytase was optimally active at pH 2.5 and 60 °C and retained activity over a wide range of temperature (40–80 °C) and pH (2.0–6.0) with a half-life of 11.3 min at 80 °C. The kinetic parameters K _m, V _max, K _cat, and K _cat/K _m of the pure phytase were 0.21 mM, 131.58 nmol mg^?1 s^?1, 1.64 × 10³ s^?1, and 7.81 × 10⁶ M^?1 s^?1, respectively. The enzyme was fairly stable in the presence of pepsin under physiological conditions. It was stimulated by Ca⁺², Mg⁺² and Mn⁺², but inhibited by Zn⁺², Cu⁺², Fe⁺², Pb⁺², Ba⁺² and surfactants. The enzyme can be applied in dephytinizing animal feeds, and the baking industry.     

Temperature and pH optima of extremely halophilic archaea: a mini-review

Archaeal microorganisms that grow optimally at Na⁺ concentrations of 1.7 M, or the equivalent of 10% (w/v) NaCl, and greater are considered to be extreme halophiles. This review encompasses extremely halophilic archaea and their growth characteristics with respect to the correlation between the extent of alkaline pH and elevated temperature optima and the extent of salt tolerance. The focus is on poly-extremophiles, i.e., taxa growing optimally at a Na⁺ concentration at or above 1.7 M (approximately 10% w/v NaCl); alkaline pH, at or above 8.5; and elevated temperature optima, at or above 50°C. So far, only a very few extreme halophiles that are able to grow optimally under alkaline conditions as well as at elevated temperatures have been isolated. The distribution of extremely halophilic archaea growing optimally at 3.4 M Na⁺ (approximately 20% w/v NaCl) is bifurcated with respect to pH optima, either they are neutrophilic, with a pH_opt of approximately 7, or strongly alkaliphilic, with pH_opt at or above 8.5. Amongst these extreme halophiles which have elevated pH optima, only four taxa have an optimum temperature above 50°C: Haloarcula quadrata (52°C), Haloferax elongans (53°C), Haloferax mediterranei (51°C) and Natronolimnobius ‘aegyptiacus’ (55°C).     

Hydrolysis of adenylyl imidodiphosphate in the presence of Na+ + Mg+ by (Na+ + K+)-activated ATPase

(1) Contrary to what has usually been assumed, (Na⁺ + K⁺)-ATPase slowly hydrolyses AdoPP[NH]P in the presence of Na⁺ + Mg²⁺ to ADP-NH₂ and P_i. The activity is ouabain-sensitive and is not detected in the absence of either Mg²⁺ or Na²⁺. The specific activity of the Na⁺ + Mg²⁺ dependent AdoPP[NH]P hydrolysis at 37°C and pH 7.0 is 4% of that for ATP under identical conditions and only 0.07% of that for ATP in the presence of K⁺. The activity is not stimulated by K⁺, nor can K⁺ replace Na⁺ in its stimulatory action. This suggests that phosphorylation is rate-limiting. Stimulation by Na⁺ is positively cooperative with a Hill coefficient of 2.4; half-maximal stimulation occurs at 5–9 mM. The K_m value for AdoPP[NH]P is 17 μM. At 0°C and 21°C the specific activity is 2 and 14%, respectively, of that at 37°C. AMP, ADP and AdoPP[CH₂]P are not detectably hydrolysed by (Na⁺ + K⁺)-ATPase in the presence of Na⁺ + Mg²⁺. (2) In addition, AdoPP[NH]P undergoes spontaneous, non-enzymatic hydrolysis at pH 7.0 with rate constants at 0, 21 and 37°C of 0.0006, 0.006 and 0.07 h^?1, respectively. This effect is small compared to the effect of enzymatic hydrolysis under comparable conditions. Mg²⁺ present in excess of AdoPP[NH]P reduces the rate constant of the spontaneous hydrolysis to 0.005 h^?1 at 37°C, indicating that the MgAdoPP[NH]P complex is virtually stable to spontaneous hydrolysis, as is also the case for its enzymatic hydrolysis. (3) A practical consequence of these findings is that AdoPP[NH]P binding studies in the presence of Na⁺ + Mg²⁺ with enzyme concentrations in the mg/ml range are not possible at temperatures above 0°C. On the other hand, determination of affinity in the (Na⁺ + K⁺)-ATPase reaction by competition with ATP at low protein concentrations (μg/ml range) remains possible without significant hydrolysis of AdoPP[NH]P even at 37°C.     

Biochemical studies of the excitable membrane of Paramecium aurelia. I. 45Ca2+ fluxes across resting and excited membrane

The characteristics of Ca²⁺ transport across the excitable membrane of Paramecium aurelia were studied by measuring ⁴⁵Ca²⁺ influx and efflux. The intracellular concentration of free Ca²⁺ in resting P. aurelia was at least ten times less than the extracellular concentration. Ca²⁺ influx was easily measurable at 0°C, but not at 23°C. The influx of ⁴⁵Ca²⁺ was stimulated by the same conditions which cause membrane depolarization and ciliary reversal. Addition of Na⁺ and K⁺ (which stimulate ciliary reversal) resulted in a 10-fold increase in the rate of Ca²⁺ influx. An externally applied, pulsed, electric field (1–2 mA/cm² of electrode surface), caused the rate of Ca²⁺ influx to increase 3–5 times, with the extent of stimulation dependent on the current density and the pulse width Ca²⁺ influx had the characteristics of a passive transport system and was associated with the chemically or electrically triggered Ca²⁺ “gating” mechanism, which has been studied electrophysiologically. In contrast, Ca²⁺ efflux appeared to be catalyzed by an active transport system. With cells previously loaded at 0°C with ⁴⁵Ca²⁺, Ca²⁺ efflux was rapid at 23°C, but did not occur at 0°C. This active Ca²⁺ efflux mechanism is probably responsible for maintaining the low internal Ca²⁺ levels in unstimulated cells.     

Equilibrium constants under physiological conditions for the reactions of the nonphosphorylated pathway of L-serine biosynthesis

The observed equilibrium constants (K_obs) for the reactions of d-2-phosphoglycerate phosphatase, d-2-Phosphoglycerate^3? + H₂O → d-glycerate^? + HPO₄^2?; d-glycerate dehydrogenase (EC 1.1.1.29), d-Glycerate^? + NAD⁺ → NADH + hydroxypyruvate^? + H⁺; and l-serine:pyruvate aminotransferase (EC 2.6.1.51), Hydroxypyruvate^? + l-H · alanine^± → pyruvate^? + l-H · serine^±; have been determined, directly and indirectly, at 38 °C and under conditions of physiological ionic strength (0.25 m) and physiological ranges of pH and magnesium concentrations. From these observed constants and the acid dissociation and metal-binding constants of the substrates, an ionic equilibrium constant (K) also has been calculated for each reaction. The value of K for the d-2-phosphoglycerate phosphatase reaction is 4.00 × 10³m [ΔG⁰ = ?21.4 kJ/mol (?5.12 kcal/mol)]([H₂0] = 1). Values of K_obs for this reaction at 38 °C, [K⁺] = 0.2 m, I = 0.25 M, and pH 7.0 include 3.39 × 10³m (free [Mg²⁺] = 0), 3.23 × 10³m (free [Mg²⁺] = 10^?3m), and 2.32 × 10³m (free [Mg²⁺] = 10^?2m). The value of K for the d-glycerate dehydrogenase reaction has been determined to be 4.36 ± 0.13 × 10^?13m (38 °C, I = 0.25 M) [ΔG⁰ = 73.6 kJ/mol (17.6 kcal/mol)]. This constant is relatively insensitive to free magnesium concentrations but is affected by changes in temperature [ΔH⁰ = 46.9 kJ/mol (11.2 kcal/mol)]. The value of K for the serine:pyruvate aminotransferase reaction is 5.41 ± 0.11 [ΔG⁰ = ?4.37 kJ/mol (?1.04 kcal/mol)] at 38 °C (I = 0.25 M) and shows a small temperature effect [ΔH⁰ = 16.3 kJ/ mol (3.9 kcal/mol)]. The constant showed no significant effect of ionic strength (0.06–1.0 m) and a response to the hydrogen ion concentration only above pH 8.5. The value of K_obs is 5.50 ± 0.11 at pH 7.0 (38 °C, [K⁺] = 0.2 m, [Mg²⁺] = 0, I = 0.25 M). The results have also allowed the value of K for the d-glycerate kinase reaction (EC 2.7.1.31), d-Glycerate^? + ATP^4? → d-2-phosphoglycerate^3? + ADP^3? + H⁺, to be calculated to be 32.5 m (38 °C, I = 0.25 M). Values for K_obs for this reaction under these conditions and at pH 7.0 include 236 (free [Mg²⁺] = 0) and 50.8 (free [Mg²⁺] = 10^?3m).     

Active vanadate-sensitive H+ translocation in corn roots membrane vesicles and proteoliposomes

A member fraction from corn roots which contains a vanadate-sensitive ATPase activity has been prepared. The specific activity at 38°C is between 3 and mol 12 μmol · min⁻¹ · mg⁻¹, depending on the age of roots. Addition of ATP promotes a very rapid quenching of the fluorescence of 9-amino-6-chloro-3-methoxy-acridin (ACMA). Proton pumping exhibits a delayed sensitivity to vanadate but is strongly and instantaneously inhibited by the new inhibitor SW 26. Both proton pumping, measured by the initial quenching rate, and ATP hydrolysis show maximum activities at ATP concentrations in the millimolar range, but the apparent K_m-value for hydrolysis is higher than that observed for proton pumping. This is interpreted as being due to the presence of two populations of ATPases, one of them hydrolyzing ATP without creating a pH-gradient. The vanadate-sensitive ATP hydrolysis and H⁺-pumping activity may be solubilized with lysolecithin and reconstituted into liposomes either by a freeze-thawing-sonication or an octylglucoside dilution procedure. Both methods yield proteoliposomes exhibiting very effecient proton pumping, which is more sensitive to vanadate (I₅₀ = 2 μM) or to SW 26 (I₅₀ = 0.5 μM) than that of the original membrane fractions.     

Activation of K-Cl Cotransport by Mild Warming in Guinea Pig Red Cells

Unidirectional, ouabain-insensitive K⁺ influx rose steeply with warming at temperatures above 37°C in guinea pig erythrocytes incubated in isotonic medium. The only component of ouabain-insensitive K⁺ influx to show the same steep rise was K-Cl cotransport (Q₁₀ of 10 between 37 and 41°C); Na-K-Cl cotransport remained constant or declined and residual K⁺ influx in hypertonic medium with ouabain and bumetanide rose only gradually. Similar results were obtained for unidirectional K⁺ efflux. Thermal activation of K-Cl cotransport-mediated K⁺ influx was fully dependent on the presence of chloride in the medium; none occurred with nitrate replacing chloride. The increase of K⁺ influx through K-Cl cotransport from 37 to 41°C was blocked by calyculin A, a phosphatase inhibitor. The Q₁₀ of K-Cl cotransport fully activated by hydroxylamine and hypotonicity was about 2. The time course of K⁺ entry showed an immediate transition to a higher rate when cells were instantly warmed from 37 to 41°C, but there was a 7-min time lag in returning to a lower rate when cells were cooled from 41 to 37°C. These results indicate that the steepness of the response of K-Cl cotransport to mild warming is due to altered regulation of the transporter. Total unidirectional K⁺ influx was equal to total unidirectional K⁺ efflux at 37–45°C, but K⁺ influx exceeded K⁺ efflux at 41°C when K-Cl cotransport was inhibited by calyculin or prevented by hypertonic incubation. The net loss of K⁺ that results from the thermal activation of isosomotic K-Cl cotransport reported here would offset a tendency for cell swelling that could arise with warming through an imbalance of pump and leak for Na⁺ or for K⁺. Received: 1 November 1997/Revised: 5 March 1998     

Acid- and temperature-induced conformational changes in human chorionic gonadotropin and the mechanism of subunit dissociation

Human chorionic gonadotropin undergoes a conformational transition in acid which at 4 °C is characterized by: (i) a reversible increase in the polarization of tyrosyl fluorescence, P, with a midpoint at pH 5, (ii) a slight decrease in the elution volume on Sephadex G-100 at pH 3 relative to pH 7, (iii) a slight decrease in s_20,w. (iv) a small positive near uv difference spectrum (Δ? ~2%), and (v) the appearance of a positive CD feature at 235 nm. These observations are compatible with an acid-expanded form of the hormone in which the rotational freedom of one or more tyrosine residues is restricted and/or their proximity to potential quenching groups is altered. The increased value of P following acidification is stable at temperatures below 10 °C, but at higher temperatures it decreases with time to an extent which is dependent on the temperature. A substantial portion of this decrease occurs before subunit dissociation can be detected, reflecting the occurrence of a thermal transition with a midpoint near 26 °C. A similar transition was observed at neutral pH with a midpoint near 22 °C. These results suggest the occurrence of at least two conformationally distinct forms of hCG which may be sequentially encountered prior to subunit dissociation in acid. The kinetics will be either biphasic or strictly first order, depending on the temperature at which the hormone is acidified.     

Effect of temperature on ion content, ion fluxes and energy metabolism in Chara corallina

Abstract Effects of temperature on the ionic relations and energy metabolism of Chara corallina were investigated. Measurements were made of the ionic content, tracer ion fluxes, and photosynthetic and dark CO₂ fixation in isolated cells, and of O₂ exchange in photosynthesis and respiration in isolated shoot apices. The total intracellular concentration of K⁺, Na⁺ and Cl^? was the same in cells held for 5 days in non-growing medium at 15°C (the growth temperature) as in those held at 25°C or 5°C. The tracer influx in the light of all ions tested (Rb⁺, Na⁺, CH₃NH₃⁺, Cl^? and H₂PO₄^?) was lower at 5°C than at 15°C in experiments in which cells were subjected to 5°C for less than 24 h in toto. The influx at 25°C was greater than that at 15°C for H₂PO^?₄, there was no difference between the two temperatures for Na⁺, while the influx at 25°C was less than that at 15°C for Cl^?, Rb⁺ and CH₃NH₃⁺ For Cl^? and H₂PO^?₄ similar results were found in later experiments with cells grown at 20—23°C. Photosynthetic CO₂ fixation and O₂ evolution, and respiratory O₂ uptake, are greater at 25°C, and lower at 5°C, than they are at the growth temperature of 15°C. In longer-term pretreatments at the different temperatures, tracer Cl^? influx at 15°C and particularly at 25°C were lower than in short-term experiments, while the influx at 5°C was higher. It was concluded from these experiments, and from previous data on H⁺ free energy differences across the plasmalemma, that (1) the maintenance of internal ion concentrations involves a close balancing of influx and efflux of K⁺, Na⁺ and Cl^? at all experimental temperatures; (2) the regulation of the tracer fluxes of the ions is kinetic rather than thermodynamic and (3) that the tracer fluxes at low temperatures are not restricted by the rate at which respiration or photosynthesis can supply energy to them.