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1.
Structural and Kinetic Properties of NADP-Malic Enzyme from the Inducible Crassulacean Acid Metabolism Plant Mesembryanthemum crystallinum L. 总被引:1,自引:0,他引:1
Saitou Kazuyuki; Agata Waichi; Asakura Masae; Kubota Fumitake 《Plant & cell physiology》1992,33(5):595-600
NADP-malic enzyme (EC 1.1.1.40
[EC]
), which is involved in Crassulaceanacid metabolism (CAM), was purified to electrophoretic homogeneityfrom the leaves of the inducible CAM plant Mesembryanthemumcrystallinum. The NADP-malic enzyme, which was purified 1,146-fold,has a specific activity of 68.8 µmol (mg protein)1min1. The molecular weight of the subunits of the enzymewas 64 kDa. The native molecular weight of the enzyme was determinedby gel-filtration to be 390 kDa, indicating that the purifiedNADP-malic enzyme is a hexamer of identical subunits. The optimalpH for activity of the enzyme was around 7.2. Double-reciprocalplots of the enzymatic activity as a function of the concentrationof L-malate yielded straight lines both at pH 7.2 and at pH7.8 and did not reveal any evidence for cooperativity of bindingof L-malate. The Km value for L-malate was 0.35 mM. Hill plotsof the activity as a function of the concentration of NADP+indicated positive cooperativity in the binding of NADP+ tothe enzyme with a Hill coefficient (nH) of 2.0. An S0.5 value(the concentration giving half-maximal activity) of 9.9 µMfor NADP+ was obtained. Oxaloacetate inhibited the activityof the NADP-malic enzyme. Effects of succinate and NaHCO3 onthe activity of NADP-malic enzyme were small. (Received October 30, 1991; Accepted May 1, 1992) 相似文献
2.
Bull R Marengo JJ Finkelstein JP Behrens MI Alvarez O 《American journal of physiology. Cell physiology》2003,285(1):C119-C128
We have reported that ryanodine receptor (RyR) channels display three different responses to cytoplasmic free Ca2+ concentration ([Ca2+]) depending on their redox state (Marengo JJ, Hidalgo C, and Bull R. Biophys J 74: 12631277, 1998), with low, moderate, and high maximal fractional open times (Po). Activation by ATP of single RyR channels from rat brain cortex was tested in planar lipid bilayers with 10 or 0.1 µM cytoplasmic [Ca2+]. At 10 µM [Ca2+], low-Po channels presented lower apparent affinity to activation by ATP [[ATP] for half-maximal activation (KaATP) = 422 µM] than moderate-Po channels (KaATP = 82 µM). Oxidation of low-Po channels with thimerosal or 2,2'-dithiodipyridine (DTDP) gave rise to moderate-Po channels and decreased KaATP from 422 to 82 µM. At 0.1 µM cytoplasmic [Ca2+], ATP induced an almost negligible activation of low-Po channels. After oxidation to high-Po behavior, activation by ATP was markedly increased. Noise analysis of single-channel fluctuations of low-Po channels at 10 µM [Ca2+] plus ATP revealed the presence of subconductance states, suggesting a conduction mechanism that involves four independent subchannels. On oxidation the subchannels opened and closed in a concerted mode. subconductance states; calcium ion release channels; calcium ion regulation; thimerosal; 2,2'-dithiodipyridine 相似文献
3.
Salinity-induced Malate Accumulation in Chara 总被引:3,自引:0,他引:3
Ion absorption by Chara corallina from solutions containingpredominantly KC1 or RbCl at up to 100 mol m3 resultedin accumulation of salts and turgor regulation. Turgor regulationdid not occur in solutions containing Na+ or Li+salts. Duringion absorption from various salts of K+ and Rb+ vacuolar cationconcentration exceeded Cl concentration. This differencewas shown to be balanced by the synthesis and accumulation ofmalate. Vacuolar malate concentration reached 48 mol m3,with accumulation occurring at rates of up to 0.45 mol m3h1. Malate accumulation was inhibited by low externalpH and was dependent upon external HCO3 concentration.The synthesis of malic acid and its subsequent dissociationimposed a severe acid load on the cell. Biophysical regulationof cellular pH was achieved by a H+efflux at a rate of about40 nmol m2 s1from the cell. The results presentedargue against cytoplasmic Cl, HCO3 or pH regulatingmalate accumulation in Chara and it is suggested that malatetransport across the tonoplast may regulate malate accumulation. Key words: Malate, Chara corallina, pH regulation, salinity 相似文献
4.
The effects of the growth in a medium containing NH4NO3 as nitrogensource were studied on cell sap pH, cytoplasmic pH and malatecontent in chl1, an Arabidopsis thaliana mutant impaired inchlorate and nitrate transport. In all the conditions testedthe pH of the cytoplasm in chl1 was more alkaline, and thatof the vacuole was more acidic as compared with those measuredin wt. Treatment with bafilomycin A1, a specific inhibitor ofthe vacuolar H+-ATPase, induced a small alkalinization of thevacuole, and a significant acidification of the cytoplasm, theseeffects being greater in chl1 than in wt. The greater responseof the mutant to bafilomycin Al suggests that, in the absenceof the inhibitor, the activity of the tonoplast H+-ATPase inchl1 is higher than in wt, this diversity being a possible reasonfor the differences in intracellular pH detected between thetwo strains. A possible role for the vacuolar H+-ATPase in regulatingthe cytoplasmic pH is discussed. (Received August 2, 1995; Accepted February 1, 1996) 相似文献
5.
MEMON ABDUL RAZAQUE; SIDDIQI M. YAEESH; CLASS ANTHONY D. M. 《Journal of experimental botany》1985,36(1):79-90
Barley (Hordeum vulgare L.) varieties differed in their raponseto [K+]0, in terms of their utilization efficiencies (UE = freshweight. concentration of [K+]11). At low [K+]0, Compana,an efficient-non-responder demonstrated superior utilizationof absorbed K+. On the other hand, at high [K+]0, Fergus (anefficient responder) and BT 334 (an inefficient responder) hadhigher UE values for K+ than Compana which performed poorlyat this [K+]0. Kinetic parameters for K+ activation of the enzyme pyruvatekinase from 12 barley varieties, representing a range of UEvalues, were determined. Varieties showed substantial differencesin their Vmax values (P<0·01). Compana, an efficientvariety, had the highest Vmax (31 µmol g1 freshwt. h1) which was about 50% higher than that of Mingo,an inefficient variety. By contrast, Km values for the enzymeswere not significantly different among varieties The mean valuesfor all varieties (3·9±0·15 mol m3K+) is far below the estimated cytoplasmic [K+] (100-200 molm3). It is, therefore, unlikely that differences in theutilization of K+ by these varieties can be explained on thebasis of differential requirements for (K+) activation of theseenzymes. Alternative possibilities for differences in the utilizationof K+ are discussed. Key words: K+ utilization efficiency, Pyruvate kinase, Barley varieties 相似文献
6.
Nitric oxide induces [Ca2+]i oscillations in pituitary GH3 cells: involvement of IDR and ERG K+ currents 总被引:1,自引:0,他引:1
Secondo A Pannaccione A Cataldi M Sirabella R Formisano L Di Renzo G Annunziato L 《American journal of physiology. Cell physiology》2006,290(1):C233-C243
The role of nitric oxide (NO) in the occurrence of intracellular Ca2+ concentration ([Ca2+]i) oscillations in pituitary GH3 cells was evaluated by studying the effect of increasing or decreasing endogenous NO synthesis with L-arginine and nitro-L-arginine methyl ester (L-NAME), respectively. When NO synthesis was blocked with L-NAME (1 mM) [Ca2+]i, oscillations disappeared in 68% of spontaneously active cells, whereas 41% of the quiescent cells showed [Ca2+]i oscillations in response to the NO synthase (NOS) substrate L-arginine (10 mM). This effect was reproduced by the NO donors NOC-18 and S-nitroso-N-acetylpenicillamine (SNAP). NOC-18 was ineffective in the presence of the L-type voltage-dependent Ca2+ channels (VDCC) blocker nimodipine (1 µM) or in Ca2+-free medium. Conversely, its effect was preserved when Ca2+ release from intracellular Ca2+ stores was inhibited either with the ryanodine-receptor blocker ryanodine (500 µM) or with the inositol 1,4,5-trisphosphate receptor blocker xestospongin C (3 µM). These results suggest that NO induces the appearance of [Ca2+]i oscillations by determining Ca2+ influx. Patch-clamp experiments excluded that NO acted directly on VDCC but suggested that NO determined membrane depolarization because of the inhibition of voltage-gated K+ channels. NOC-18 and SNAP caused a decrease in the amplitude of slow-inactivating (IDR) and ether-à-go-go-related gene (ERG) hyperpolarization-evoked, deactivating K+ currents. Similar results were obtained when GH3 cells were treated with L-arginine. The present study suggests that in GH3 cells, endogenous NO plays a permissive role for the occurrence of spontaneous [Ca2+]i oscillations through an inhibitory effect on IDR and on IERG. voltage-gated potassium channels; ether-à-go-go-related gene potassium channels; slow-inactivating outward currents; fast-inactivating outward currents 相似文献
7.
The turgor regulation induced by hypotonic treatment (hypotonicturgor regulation) in the brackish-water alga Lamprothamniumsuccinctum is accompanied by a transient increase in the electricalconductance of the membrane, membrane depolarization and a transientincrease in the cytoplasmic concentration of free Ca2+ ([Ca2+([Ca2+]c) (Okazaki and Tazawa 1990). In the present study, weloaded a Ca2+-chelating agent, EGTA, into the cytoplasm by mechanicalinjection in order to suppress the increase in [Ca2+]c thatoccurs during the hypotonic turgor regulation. The rate of thecytoplasmic streaming was taken as an indirect indicator of[Ca2+]c, since cytoplasmic streaming has been shown to be inhibitedby high [Ca2+]c in Lamprothamnium cells. The lag time for theinhibition of the cytoplasmic streaming upon hypotonic treatmentwas significantly prolonged in EGTA-loaded cells as comparedto that in intact cells. This result indicates that the loadedcytoplasmic EGTA functioned as a buffer of Ca2+ to retard theincrease in [Ca2+]c. It took a longer time for the membraneconductance to reach the peak value in EGTA-loaded cells thanin intact cells. Membrane depolarization was affected to aninsignificant extent by the cytoplasmic EGTA. The regulationof turgor pressure itself was partially inhibited. These resultsstrongly support the idea that the net efflux of ions that occursduring the recovery from hy-potonically induced changes in turgorpressure is controlled by [Ca2+]c. (Received August 22, 1990; Accepted December 6, 1990) 相似文献
8.
Using permeabilized characean cells in which the ionic conditionsat the cytoplasmic side of the tonoplast are easily controlled,effects of Ca2+ ion on tonoplast potential were examined. Whenthe cell was treated with 1 µM Ca2+, the tonoplast potential(EM became positive in a complicated manner in Chara corallinawhile it simply became negative in Nitella axilliformis. Whenthe cell was treated with 9-antracenecarboxylic acid, a Cl-channelinhibitor, Em became more negative and the response of Em toCa2+ was significantly suppressed. It is suggested that Ca2+activates Cl-channel at a low concentration and inactivatesat a higher one in C. corallina while it simply inactivate Cl-channelin N. axilliformis.
1Present address: Biological Laboratory, The University of theAir, Wakaba 2-11, Wakaba, 260 Japan. (Received August 22, 1988; Accepted December 26, 1988) 相似文献
9.
Proton Transport and pH Control in Sinapis alba Root Hairs: A Study carried out with Double-Barrelled pH Micro-electrodes 总被引:3,自引:0,他引:3
Continuous measurements of cytoplasmic pH (pHc) in Sinapis roothairs have been carried out with double-barrelled pH-micro-electrodesin order to gain information on translocation of protons acrossthe plasmalemma and cytoplasmic pH control. (i) The cytoplasmicpH of Sinapis (733 ? 012, standard conditions)changes no more than 0.1 pHc, per pHo-unit, regardless of whethercyanide is present or not. (ii) Weak acids rapidly acidify pHcand hyperpolarize, while weak bases alkalize pHc and depolarizethe cells, (iii) 1.0 mol M,3 NaCN acidifies the cytoplasm by0.4 to 0.7 pH-units, but alkalizes the vacuole. (iv) 20 mmolm3 CCCP has no significant effect on pHc, if added atpH 9.6 or 7.2, but acidifies pHc by 1.3 units at pH 4.3. Inthe presence of CCCP, cyanide acidifies the cytoplasm, (v) Chloridetransiently acidifies pHc, while K+, Na+, and have no significant effects, (vi) Cytoplasmic buffer capacityforms a bell-shaped curve versus pHc with an optimum of about50 mol m3 H+pHc-unit. The modes of proton re-entry and the effects of active and passiveproton transport on cellular pH control are critically discussed.It is suggested that the proton leak, consisting of H+-cotransport(e.g. H+/Cl) rather than H+-uniport, is no threat topHc. The proton export pump, although itself reacting to changesin pHc, influences pHc only to a minor extent. It is concludedthat buffer capacity and membrane transport play moderate rolesin pHc control in Sinapis, while the interlocked H+-producingand -consuming reactions of cellular metabolism are the mainregulating factors. This makes pH control in Sinapis quite differentfrom bacterial and animal cells. Key words: Cytoplasmic pH, double-barrelled pH micro-electrode, pH control, proton transport, Sinapis 相似文献
10.
Carbonic anhydrase (CA, EC. 4.2.1.1
[EC]
) activity in air-grown Characorallina was detected mainly in the intracellular fraction,most of which composed of chloroplasts and cytoplasmic gel,and not on the cell surface. Only minor levels of CA activity,on the basis of equivalent volumes, were detected in the cellsap and the cytoplasmic sol. The maximum rate of photosynthetic O2 evolution by air-grownChara corallina at pH 6.0 was twice that at pH 7.6, while theapparent Km for external inorganic carbon (Ci) at pH 7.6 wasabout three times that at pH 6.0. However, the apparent Km(CO2)was about three times larger at pH 6.0 than at pH 7.6. The Km(Ci)-valueat pH 7.6 increased severalfold in the presence of acetazolamide(AZA), an inhibitor of CA, but no inhibition was observed atpH 6.0. The pH-dependence may be due to differences in the permeabilityof AZA at the given pH values. Fixation of 14CO2 at 20 µMand of H14CO3 at 200 µM over the course of 5 swas very similar at pH 7.4. Addition of CA significantly suppressedthe photosynthetic 14CO2-fixation but it stimulated the H14CO3-fixation.This result indicates that free CO2 is an active species ofCi that is incorporated into the cell during photosynthesis. These results together suggest the following: (1) Free CO2 isutilized for photosynthesis, (2) CA is mainly located insidethe cell and functions to increase the affinity for CO2 in photosynthesisby facilitating the supply of CO2 from the plasmalemma to thesite of CO2-fixation.
3Present address: Biological Laboratory, The University of theAir, Wakaba 2-11, Chiba, 260 Japan. (Received December 9, 1988; Accepted March 22, 1989) 相似文献
11.
Yazaki Yoshiaki; Asukagawa Norio; Ishikawa Yutaka; Ohta Eiji; Sakata Makoto 《Plant & cell physiology》1988,29(6):919-924
The cytoplasmic [MgATP]/[ATP]free ratios, free Mg2+ concentrations,and phosphorylation potentials in mung bean [Vigna mungo (L.)Hepper] root tip cells were investigated by 31P nuclear magneticresonance spectroscopy. 31P NMR spectra show well defined peaksdue to G6P, cytoplasmic Pi, vacuolar Pi, ATP, UDP-glucose andnicotinamide adenine nucleotides. The concentrations of phosphorusmetabolites were determined from quantitative 31P NMR spectra.The [MgATP]/[ATP]free ratio was 9.45. Accordingly, about 90%of the cytoplasmic ATP was complexed to Mg2+. Utilizing thedissociation constant (Kd) determined for MgATP, the cytoplasmicfree Mg2+ concentration was estimated to be 0.4mM. The NMR-derivedphosphorylation potential, [ATP]/([ADP][Pi]), was 960 M-1. Thesodium azide treatment decreased the [ATP]/[ADP] ratio and thephosphorylation potential, and increased the [Mg2+]free. Metabolicinhibition may have been enhanced by an increase in [Mg2+freeand a decrease in the free energy change for ATP hydrolysis,which resulted due to a decrease in the ATP level.
1Present address: National Food Research Institute, TsukubaCity, Ibaraki 305, Japan. (Received February 8, 1988; Accepted June 1, 1988) 相似文献
12.
Efficiency of Potassium Utilization by Barley Varieties: The Role of Subcellular Compartmentation 总被引:6,自引:3,他引:3
RAZAQUE MEMON ABDUL; SACCOMANI MASSIMO; GLASS ANTHONY D. M. 《Journal of experimental botany》1985,36(12):1860-1876
Memon, A. R., Saccomani, M. and Glass, A. D. M. 1985. Efficiencyof potassium utilization by barley varieties: The role of subcellularcompartmentation.?J. exp. Bot. 36: 18601876. The subcellulardistributions of K+ in roots of three barley (Hordeum vulgareL.) varieties, grown at 10 and 100 mmol m3 external K+([K+]o) were estimated by compartmental analyses. In general,increased [K+]o caused a 23 fold increase in vacuolar[K+], but cytoplasmic [K+] increased only slightly. Nevertheless,the three varieties, which had been selected for study on thebasis of their different rates of K+ utilization, showed distinctdifferences in the allocation of K+ between cytoplasm and vacuole.At 10 mmol m3 [K+]o var. Betzes exhibited typical K+deficiency symptoms while var. Fergus and var. Compana did not,even though Betzes had higher [K+] in shoots and roots. Theinefficient utilization of K+ in this variety appears to beassociated with a failure to mobilize vacuolar K+ into the cytoplasmiccompartment (the ratio of vacuolar: cytoplasmic K+ contentsfor Betzes was 4.1 compared to 2.7 and 2.5, respectively, forFergus and Compana). Fergus and Betzes, which demonstrate pronouncedgrowth responses to increased [K+]0 between 10 and 100 mmolm3, showed significant increases of cytoplasmic [K+]in this range of [K+]o. By contrast, cytoplasmic [K+] in Compana,a variety whose growth is not stimulated by increased [K+]0(from 10 to 100 mmol m3) showed virtually no increase.It is suggested that the efficiency of K+ utilization and thegrowth response to [K+]0 in these varieties are functions ofthe subcellular distribution of this ion between cytoplasm andvacuole. Key words: Barley varieties, K+ subcellular compartmentation, utilization efficiency 相似文献
13.
Effects of cytoplasmic Ca2+ on the electrical properties ofthe plasma membrane were investigated in tonoplast-free cellsof Chara australis that had been internally perfused with media,containing either 1 mM ATP to fuel the electrogenic pump orhexokinase and glucose to deplete the ATP and stop the pump. In the presence of ATP, cytoplasmic Ca2+ up to 2.5?105M did not affect the membrane potential (about -190 mV), butmembrane resistance decreased uniformly with increasing [Ca2+]i.In the absence of ATP, the membrane potential, which was onlyabout -110 mV, was depolarized further by raising [Ca2+]i from1.4?106 to 2.5?105 M. Membrane resistance, whichwas nearly the twofold that of ATP-provided cells, decreasedmarkedly with an increase in [Ca2+]i from zero to 1.38?106M, but showed no change for further increases. Internodal cellsof Nitellopsis obtusa were more sensitive to intracellular Ca2+with respect to membrane potential than were those of Charaaustralis, reconfirming the results obtained by Mimura and Tazawa(1983). The effect of cytoplasmic Ca2+ on the ATP-dependent H+ effluxwas measured. No marked difference in H+ effluxes was detectedbetween zero and 2.5?105 M [Ca2+]i; but, at 104M the ATP-dependent H+ efflux was almost zero. Ca2+ efflux experimentswere done to investigate dependencies on [Ca2+]i and [ATP]i.The efflux was about 1 pmol cm2 s1 at all [Ca2+]iconcentrations tested (1.38?106, 2.5?105, 104M).This value is much higher than the influx reported by Hayamaet al. (1979), and this efflux was independent of [ATP]i. Thepossibility of a Ca2+-extruding pump is discussed.
1 Present address: Botanisches Institut der Universit?t Bonn,Venusbergweg 22, 5300 Bonn, F.R.G. (Received September 22, 1984; Accepted February 19, 1985) 相似文献
14.
Torimitsu Keiichi; Yazaki Yoshiaki; Nagasuka Kinuyo; Ohta Eiji; Sakata Makoto 《Plant & cell physiology》1984,25(8):1403-1409
The effect of the external pH on the intracellular pH in mungbean (Vigna mungo (L.) Hepper) root-tip cells was investigatedwith the 31P nuclear magnetic resonance (NMR) method. The 31PNMR spectra showed three peaks caused by cytoplasmic G-6-P,cytoplasmic Pi and vacuolar Pi. The cytoplasmic and vacuolarpHs could be determined by comparing the Pi chemical shiftswith the titration curve. When the external pH was changed overa range from pH 3 to 10, the cytoplasmic pH showed smaller changesthan the vacuolar pH, suggesting that the former is regulatedmore strictly than the latter. The H+-ATPase inhibitor, DCCD,caused the breakdown of the mechanism that regulates the intracellularpH. H+-ATPase appears to have an important part in the regulationof the intracellular pH. (Received January 4, 1984; Accepted August 27, 1984) 相似文献
15.
DiPolo R Berberián G Beaugé L 《American journal of physiology. Cell physiology》2000,279(5):C1631-C1639
We investigated the role of intracellular Mg2+(Mgi2+) on the ATP regulation ofNa+/Ca2+ exchanger in squid axons and bovineheart. In squid axons and nerve vesicles, the ATP-upregulated exchangerremains activated after removal of cytoplasmic Mg2+, evenin the absence of ATP. Rapid and complete deactivation of theATP-stimulated exchange occurs upon readmission ofMgi2+. At constant ATP concentration, the effectof intracellular Mg2+ concentration([Mg2+]i) on the ATP regulation of exchangeris biphasic: activation at low [Mg2+]i,followed by deactivation as [Mg2+]i isincreased. No correlation was found between the above results and thelevels of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] measured innerve membrane vesicles. Incorporation ofPtdIns(4,5)P2 into membrane vesicles activates Na+/Ca2+ exchange in mammalian heart but not insquid nerve. Moreover, an exogenous phosphatase prevents MgATPactivation in squid nerves but not in mammalian heart. It is concludedthat 1) Mgi2+ is an essentialcofactor for the deactivation part of ATP regulation of the exchangerand 2) the metabolic pathway of ATP upregulation of theNa+/Ca2+ exchanger is different in mammalianheart and squid nerves. 相似文献
16.
RuDP carboxylase isolated from autotrophically grown cells ofphotosynthetic sulfur bacterium, Chromatium strain D, was partiallypurified by (NH4)2SO4 precipitation and Sephadex G-200 gel filtration.The molecular size of the bacterial RuDP carboxylase was foundto be large, analogous to that of the plant enzyme, in agreementwith results of previous workers. Sucrose density gradient centrifugationshowed the Srel to be approximately 18; the omission of Mg++caused no dissociation of the enzyme molecule in its subunits.Chromatium RuDP carboxylase showed similarities to the plantenzyme in some of its kinetic properties; (a) a shift of pHoptimum to the neutral side from the alkaline side on the additionof Mg++, (b) deviation of the substrate concentration (NaHCO3)-activityrelationship from the MICHAELIS formula and (c) a marked stimulativeeffect of Mg++. A unique sigmoidal saturation curve of the enzymeto RuDP, which had been detected in Rhodospirillum rubrum andRhodopseudomonas spheroides RuDP carboxylase in the absenceof Mg++, was not found. Another characteristic feature of ChromatiumRuDP carboxylase is its partial immunological response to therabbit anti-spinach RuDP carboxylase serum as detected by theinhibition of the carboxylation reaction due to the antibody-antigenreaction.
1Part X, Structure and Function of Chloroplast Proteins. Supportedin part by research grants from the Ministry of Education ofJapan (No. 8719) and USPHS (AM-10792-03) (Received July 4, 1969; ) 相似文献
17.
Mujer Cesar V.; Fox Theodore C.; Williams Adrienne S.; Andrews David L.; Kennedy Robert A.; Rumpho Mary E. 《Plant & cell physiology》1995,36(8):1459-1470
Enolase (2-phospho-D-glycerate hydrolyase, EC 4.2.1.11
[EC]
) activityis differentially induced by anoxia in the flood-tolerant speciesE. phyllopogon (Stev.) Koss and the flood-intolerant speciesE. crus-pavonis (H.B.K.) Schult. To examine the regulation ofenolase at the protein level, we purified the enzyme from bothspecies to near homogeneity and compared their physico-chemicaland catalytic properties. Enolase purified from E. phyllopogonexhibits optimal activity at pH 7.0, a Km of 80 µM for2-PGA, a Q10 of 1.97 and an Ea of 12.3 kcal mol-1. Similarly,enolase from E. crus-pavonis exhibits optimal activity at pH7.0, a Km of 50 µM for 2-PGA, a Q10 of 2.04 and an Eaof 12.9 kcal mol-1. The enzyme from both species is thermostable(100% active after 15 min, 50°C) and is a homodimer of 52.5kDa subunits as resolved by SDS-PAGE and immunoblotting. E.phyllopogon enolase was phosphorylated in vitro using either[ 相似文献
18.
Cytoplasmic pH (pHc) in Chara corallina was measured (from [14C]stribution)as a function of external pH (pH0)and temperature. With pH0near 7, pHc at 25?C is 7.80; pHcincreases by 0.005 pH units?C1 temperature decrease, i.e. pHc at 5 ?C is 7.90. WithpH? near 5.5, the increase in pHc with decreasing temperatureis 0.015 units ?C1 between 25 and 15?C, but 0.005 units?C1 between 15 and 5?C. This implies a more precise regulationof pHc with variations in pHo at 5 or 15 ?C compared with 25?C. The observed dp Hc/dT is generally smaller than the 0.017units ?C1 needed to maintain a constant H+/OH1,or a constant fractional ionization of histidine in protein,with variation in temperature. It is closer to that needed tomaintain the fractional ionization of phosphorylated compoundsor of CO2HCO3 The value of dpHc/dT has importantimplications for several regulatory aspects of cell metabolism.These include (all as a function of temperature) the rates ofenzyme reactions, the H+ at the plasmalemma(and hence the energy available for cotransport processes),and the mechanism for pHc regulation by the control of bidirectionalH+ fluxes at the plasmalemma. 相似文献
19.
H+ translocation driven by NO3, NO2 and N2O reductionswith endogenous substrates in cells of Rhodopseudomonas sphaeroidesforma sp. denitrificans was investigated by the oxidant pulsemethod. Upon injection of nitrogenous oxides to anaerobic cellsin darkness, an alkaline transient in the external medium wasobserved, followed by acidification. The alkaline transientwas enhanced by carbonyl cyanide m-chlorophenylhydrazone. When a viologen dye was used as an electron donor in the presenceof 1 mM Af-ethylmaleimide and 0.1 mM 2-n-heptyl-4-hydroxyquinoline-N-oxideto preclude respiration-linked H+ extrusion, addition of KNO3,KNO2 and N2O caused only a rapid alkalinization. The H+ consumptionstoichiometries, H+/2e ratios for NO3 reductionto NO2, NO2 reduction to 1/2 N2O and N2O reductionto N2 were 1.90, 3.18 and 2.04, respectively.These values agreed well with the fact that all reductions ofnitrogenous oxides in denitrification occur on the periplasmicside of the cytoplasmic membrane. When corrected for H+ consumption in the periplasm, the H+ extrusionstoichiometries, H+/2e ratios with endogenous substratesin the presence of K+/valinomycin for NO3 reduction toNO2, NO2 reduction to 1/2 N2O and N2O reductionto N2 were 4.05, 4.95 and 6.01, respectively. (Received August 4, 1982; Accepted January 13, 1983) 相似文献
20.
Using the compartmental analysis the unidirectional Na+ fluxesin cortical cells of barley roots, the cytoplasmic and vacuolarNa+ contents Qc and Qv, and the trans-root Na+ transport R'have been studied as a function of the external Na+ concentration.Using the re-elution technique the effect of low K+ concentrationson the plasmalemma efflux co of Na+ (K+-Na+ exchange) and onR' was investigated at different Na+ concentrations and correspondinglydifferent values of the cytoplasmic sodium content Qc. The relationof the K+-dependent Na+ efflux coK+-dep to Qc or to the cytoplasmicNa+ concentration obeyed Michaelis-Menten kinetics. This isconsistent with a linkage of co, K+-dep to K+ influx by a K+-Na+exchange system. The apparent Km corresponded to a cytoplasmicNa+ concentration of 28 mM at 0·2 mM K+ and about 0·2mM Na+ in the external solution. 0·2 mM K+ stimulatedthe plasma-lemma efflux of Na+ and inhibited Na+ transport selectivelyeven in the presence of 10 mM Na+ in the external medium showingthe high efficiency of the K+-Na+ exchange system. However,co, K+-dep was inhibited at 10 mM Na1 compared to lower Na1concentrations suggesting some competition of Na1 with K1 atthe external site of the exchange system. The effect of theNa+ concentration on Na1 influx oc is discussed with respectto kinetic models of uuptake. 相似文献