Proton transport is often visualized in membrane vesicles by use of fluorescent monoamines which accumulate in acidic intravesicular compartments and undergo concentration-dependent fluorescence quenching. Software for an IBM microcomputer is described which permits logging and editing of changes in fluorescence monitored by a Perkin-Elmer LS-5 luminescence spectrometer. An accurate estimate of the instantaneous rate of fluorescence quenching or recovery is then facilitated by least squares fitting of fluorescence data to a nonlinear function. The software is tested with tonoplast vesicles from Beta vulgaris. Quenching of acridine orange fluorescence by ATP-driven (primary) transport and relaxation of quenching by Na+/H+ antiport can both be fitted with single exponential functions. Initial rates of ATP- and Na+ -dependent fluorescence changes are derived and can be used for Km determinations. The method constitutes a simple and efficient alternative to manual analysis of analog fluorescence traces and results in a reliable quantitative measurement of the relative rate of proton transport in membrane vesicle preparations.
The permeability of mitochondria from pea (Pisum sativum L. var Kleine Rheinländerin) leaves, etiolated pea shoots, and potato (Solanum tuberosum) tuber for malate, oxaloacetate, and other dicarboxylates was investigated by measurement of mitochondrial swelling in isoosmolar solutions of the above mentioned metabolites. For the sake of comparison, parallel experiments were also performed with rat liver mitochondria. Unlike the mammalian mitochondria, the plant mitochondria showed only little swelling in ammonium malate plus phosphate media but a dramatic increase of swelling on the addition of valinomycin. Similar results were obtained with oxaloacetate, maleate, fumarate, succinate, and malonate. n-Butylmalonate and phenylsuccinate, impermeant inhibitors of malate transport in mammalian mitochondria, had no marked inhibitory effect on valinomycin-dependent malate and oxaloacetate uptake of the plant mitochondria. The swelling of plant mitochondria in malate plus valinomycin was strongly inhibited by oxaloacetate, at a concentration ratio of oxaloacetate/malate of 10−3. From these findings it is concluded: (a) In a malate-oxaloacetate shuttle transferring redox equivalents from the mitochondrial matrix to the cytosol, malate and oxaloacetate are each transported by electrogenic uniport...
Vein loading of exogenous [14C]sucrose was studied using short uptake and wash periods to distinguish between direct loading into veins and loading via mesophyll tissue. Mature leaf tissue of Pisum sativum L. cv Little Marvel, or Coleus blumei Benth. cv Candidum, was abraded and leaf discs were floated on [14C]sucrose solution for 1 or 2 minutes. Discs were then washed for 1 to 30 min either at room temperature or in the cold and were frozen, lyophilized, and autoradiographed. In P. sativum, veins were clearly labeled after 1 minute uptake and 1 minute wash periods. Autoradiographic images did not change appreciably with longer times of uptake or wash. Vein loading was inhibited by p-chloromercuribenzenesulfonic acid. These results indicate that uptake of exogenous sucrose occurs directly into the veins in this species. When C. blumei leaf discs were floated on [14C]sucrose for 2 minutes and washed in the cold, the mesophyll was labeled but little, if any, minor vein loading occurred. When discs were labeled for 2 minutes and washed at room temperature, label was transferred from the mesophyll to the veins within minutes. These results indicate that there may be different patterns of phloem loading of photosynthetically derived sucrose in these two species.
The relationship between steady-state photosynthetic efficiency, as moles CO2 per mole of incident visible photons under 2% O2, and chlorophyll fluorescence quenching has been investigated in intact leaf tissue of Spinacia oleracia. Fluorescence yield was measured using a pulse amplitude modulation technique that permitted rapid and sensitive resolution and quantitation of photochemical and nonphotochemical quenching coefficients. A highly linear relationship was observed between photosynthetic efficiency and the ratio of photochemical:nonphotochemical quenching coefficients for values of the latter less than 1.6. This relationship applied whether irradiance or CO2 concentration was varied. The observed relationships between photochemical yield and fluorescence yield were compatible with the photosystem II model proposed by Butler and Kitajima (1975 Biochim Biophys Acta 376: 116-125). The results are discussed with respect to the proposed role of nonphotochemical quenching in regulating radiant energy utilization and also the applicability of fluorescence measurements as a means of estimation of the rate of photosynthetic electron transport.
To further our understanding of the role of Cl− and certain other monovalent anions in the oxygen evolving photosystem II of chloroplasts, dissociating and stabilizing anion effects on the extrinsic 17 and 23 kilodalton polypeptides of the photosynthetic water oxidizing complex were investigated. It was found that (a) the dissociation of the two polypeptides in Cl− free media of pH ≈ 7 was enhanced by millimolar concentrations of the divalent anion SO42− and also by divalent cations like Mg2+ and Ca2+; (b) the dissociation was opposed by relatively low concentrations of monovalent anions with an order of effectiveness Cl− = Br− > NO3− > F− > ClO4−; (c) at molar concentrations, SO42− stabilized the binding of the 23 kilodalton polypeptide, while Cl− and Br− became dissociating agents, in agreement with studies by Blough and Sauer (1984 Biochim Biophys Acta 767: 377-381); (d) the binding of the polypeptides was strengthened at room temperature relative to 0°C, indicating an involvement of hydrophobic forces. It is suggested that a specific binding of Cl−, or certain substitutes, organizes the protein surfaces and/or the adjacent water layers in the water oxidizing complex in a way that not only stabilizes its assembly...
A kinetic analysis was made of l-valine uptake in protoplast-derived cells (mesophyll protoplasts cultured for 6 days) and in suspension-cultured cells of tobacco (Nicotiana tabacum L., cv Xanthi). Cells from wild-type and Valr-2 mutant plants were compared. A low-Km component was found in protoplast-derived cells (Km = 45 ± 5 micromolar) as well as in suspension-cultured cells (Km = 84 ± 21 micromolar). In the mutant cells the Vmax of this component was 12- to 14-fold less than in wild-type cells. A second component (Km = 2.4 ± 0.7 millimolar) was found in suspension-cultured cells but not in protoplast-derived cells; its Vmax was the same in wild-type and mutant cells. A third component was apparently unsaturable (linear component). It was present in protoplast-derived cells but not in suspension-cultured cells, and had the same magnitude in wild-type and mutant cells. The results are discussed with reference to the uptake of l-valine in leaf tissue, in which the three kinetic components have been found simultaneously. The reduced Vmax of the low-Km component in the Valr-2 mutant, and the differential expression of the other two components in suspension-cultured cells and protoplast-derived cells indicate that the kinetically distinguishable components represent physically distinct transport systems.
We report here on an experimental system that utilizes ion-selective microelectrodes to measure the electrochemical potential gradients for H+ and K+ ions within the unstirred layer near the root surface of both intact 4-day-old corn seedlings and corn root segments. Analysis of the steady state H+ and K+ electrochemical potential gradients provided a simultaneous measure of the fluxes crossing a localized region of the root surface. Net K+ influx values obtained by this method were compared with unidirectional K+ (86Rb+) influx kinetic data; at any particular K+ concentration, similar values were obtained by either technique. The ionspecific microelectrode system was then used to investigate the association between net H+ efflux and net K+ influx. Although the computed H+:K+ stoichiometry is dependent upon the choice of diffusion coefficients, the values obtained were extremely variable, and net K+ influx rarely appeared to be charge-balanced by H+ efflux. In contrast to earlier studies, we found the cortical membrane potential to be highly K+ sensitive within the micromolar K+ concentration range. Simultaneous measurements of membrane potential and K+ influx, as a function of K+ concentration, revealed similar Km values for the depolarization of the potential (Km 6-9 micromolar K+) and net K+ influx (Km 4-7 micromolar K+). These data suggest that K+ may enter corn roots via a K+-H+ cotransport system rather than a K+/H+ antiporter.
The photochemical apparatus organization in the thylakoid membrane of Macrocystis pyrifera, the giant kelp, was investigated. Chloroplasts were isolated from surface and minus 20 meter blades. Photosynthetic electron-transport complex quantitation revealed ratios of photosystem (PS) II/cytochrome b6-f/PSI = 1.8:3.3:1.0 in surface and 2.2:2.3:1.0 in minus 20 meter blades. The apparent photosynthetic unit size of chloroplasts from minus 20 meter blades (chlorophyll/P700 = 1485:1) was about 45% larger than that of surface blades (chlorophyll/P700 = 1025:1). The larger photosynthetic unit size of minus 20 meter blades is attributed to the substantially lower intensity of sunlight reaching the minus 20 meter habitat. In different chloroplast preparations, the effective absorption cross section of PSI and PSII to 670 nanometer light (chlorophyll a) and 481 nanometer light (chlorophyll c and fucoxanthin) was investigated. The results showed larger functional antenna size for PSII (about 90%) and for PSI (about 50%) in minus 20 meter than in surface blades. Moreover, the efficiency of utilization of 481 nanometer light by Macrocystis chloroplasts was equal to that of 670 nanometer light. It is concluded that the chlorophyll c-fucoxanthin complex in brown algae enables the highly efficient utilization of blue-green wavelengths of the nearshore marine environment and contributes to the dominance of M. pyrifera in this habitat.
Potassium influx into roots of dark-grown decapitated maize seedling (Zea mays L., cv Pioneer 3369A) was examined in presence and absence of ambient ammonium and at various root potassium concentrations. Six-day old seedlings which were dependent on the endosperm reserves for their energy source were exposed to KCl (labeled with 86Rb) ranging from 5 to 200 micromolar. At both low (13 micromoles per gram fresh weight) and high (100 micromoles per gram fresh weight) root potassium concentration, isotherms indicated two potassium influx systems, one approaching saturation at 50 to 100 micromolar potassium and an additional one tentatively considered to be linear. A mixed-type inhibition by ammonium for the low-concentration saturable system was indicated by a concomitant decrease in Vmax and increase in Km. High root potassium concentration decreased Vmax but had little effect on Km of this system. The rate constant for the second quasilinear system was decreased by ambient ammonium and by high root potassium status. Transfer of high potassium roots to potassium-free solutions resulted in an increase in influx within 2 hours; by 24 hours influx significantly exceeded that of roots not previously exposed to potassium. In roots of both low and high root potassium concentrations...
Plasma membrane preparations of high purity (about 95%) are easily obtained by partitioning in aqueous polymer two-phase systems. These preparations, however, mainly contain sealed right-side-out (apoplastic side out) vesicles. Part of these vesicles have been turned inside-out by freezing and thawing, and sealed inside-out and right-side-out vesicles subsequently separated by repeating the phase partition step. Increasing the KCI concentration in the freeze/thaw medium as well as increasing the number of freeze/thaw cycles significantly increased the yield of inside-out vesicles. At optimal conditions, 15 to 25% of total plasma membrane protein was recovered as inside-out vesicles, corresponding to 5 to 10 milligrams of protein from 500 grams of sugar beet (Beta vulgaris L.) leaves. Based on enzyme latency, trypsin inhibition of NADH-cytochrome c reductase, and H+ pumping capacity, a cross-contamination of about 20% between the two fractions of oppositely oriented vesicles was estimated. Thus, preparations containing about 80% inside-out and 80% right-side-out vesicles, respectively, were obtained. ATPase activity and H+ pumping were both completely inhibited by vanadate (Ki ≈ 10 micromolar), indicating that the fractions were completely free from nonplasma membrane ATPases. Furthermore...
(22S,23S)-Homobrassinolide was tested for its effect on the electric cell potential, proton extrusion, ferricyanide reduction, and amino acid and sucrose uptake of leaves of Egeria densa Planchon. In the light, (22S,23S)-homobrassinolide and its derivative, 2α-3α-dihydroxy-5α-stigmast-22-en-6-one, were similar to each other and similar to fusicoccin in causing hyperpolarization and proton extrusion, whereas stigmasterol was less effective. In darkness, the three sterols showed comparable effects. (22S,23S)-Homobrassinolide slightly stimulated ferricyanide reduction and promoted uptake of sucrose and α-aminoisobutyric acid. The results are compatible with a stimulation of an electrogenic proton pump mechanism at the plasmalemma by (22S,23S)-homobrassinolide.
Elongation of subapical segments of maize (Zea mays) roots was greatly inhibited by 2H2O in the incubation medium. Short-term exposure (30 min) to 2H2O slightly reduced O2 uptake and significantly increased ATP levels. 2H2O inhibited H+ extrusion in the presence of both low (0.05 mm) and high (5 mm) external concentrations of K+ (about 30 and 53%, respectively at 50% [v/v] 2H2O). Experiments on plasma membrane vesicles showed that H+-pumping and ATPase activities were greatly inhibited by 2H2O (about 35% at 50% [v/v] 2H2O); NADH-ferricyanide reductase and 1,3-β-glucan synthase activities were inhibited to a lesser extent (less than 15%). ATPase activities present in both the tonoplast-enriched and submitochondrial particle preparations were not affected by 2H2O. Therefore, the effect of short incubation time and low concentration of 2H2O is not due to a general action on overall cell metabolism but involves a specific inhibition of the plasma membrane H+ -ATPase. K+ uptake was inhibited by 2H2O only when K+ was present at a low (0.05 mm) external concentration where absorption is against its electrochemical potential. The transmembrane electric potential difference (Em) was slightly hyperpolarized by 2H2O at low K+, but was not affected at the higher K+ concentrations. These results suggest a relationship between H+ extrusion and K+ uptake at low K+ external concentration.
Pulsed, time resolved photoacoustics has sufficient sensitivity to determine oxygen emission and uptake by single turnover flashes to leaves. The advantage over previous methodologies is that when combined with single turnover flashes the kinetics of the thermal and the gas signals can be resolved to 0.1 millisecond and separated. The S-state oscillations of oxygen formation are readily observed. The gas signal from common spongy leaves such as spinach (Spinacia sp.), Japanese andromeda (Pieris japonica), mock orange (Philadelphus coronarius) and viburnum (Viburnum tomentosum), after correction for instrumental rise time, show a lag of only 1 millisecond and a rise time of 5 milliseconds in the formation of oxygen. Thus a recent proposal that the formation of oxygen requires over 100 milliseconds cannot be true for choroplasts in vivo. The rapid emission is correlated with structure of the leaf. At low light flash energies a rapid gas uptake is observed. The uptake has slightly slower kinetics than oxygen evolution, and its magnitude increases with damage to the leaf. The pulse methodology shows that the uptake begins with the very first flash after dark adaption, and allows the detection of a positive signal (oxygen) on the third flash. These observations...
Proton excretion from bean (Phaseolus vulgaris L.) leaf cells is increased by bright white light. To test whether this could be due, at least in part, to an increase in plasma membrane (PM) ATPase activity, PM vesicles were isolated from primary leaves by phase partitioning and used to characterize PM ATPase activity and changes in response to light. ATPase activity was characterized as magnesium ion dependent, vanadate sensitive, and slightly stimulated by potassium chloride. The pH optimum was 6.5, the Km was approximately 0.30 millimolar ATP, and the activity was about 60% latent. PM vesicles were prepared from leaves of plants grown for 11 days in dim red light (growing slowly) or grown for 10 days in dim red light and then transferred to bright white-light for 1 day (growing rapidly). For both light treatments, ATPase specific activity was approximately 600 to 700 nanomoles per milligram protein per minute, and the latency, Km, and sensitivity to potassium chloride were also similar. PM vesicles from plants grown in complete darkness, however, exhibited a twofold greater specific activity. We conclude that the promotion of leaf growth and proton excretion by bright white light is not due to an increase in ATPase specific activity. Light does influence ATPase activity...
The properties of the vacuolar membrane (tonoplast) ion channels of sugar beet (Beta vulgaries) cell cultures were studied using the patch-clamp technique. Tonoplast currents displayed inward rectification in the whole vacuole and isolated outside-out patch configurations and permeability ratios PK+/PNa+ = 1 and PK+/PCl− = 5. Amiloride and two of its analogs, 5-(N-methyl-N-isobutyl)-amiloride and benzamil, inhibitors of Na+ channels in animal systems, blocked inward currents by reducing single-channel openings. Concentrations for 50% inhibition of vacuolar currents of 730 nanomolar, 130 nanomolar, and 1.5 micromolar for amiloride, benzamil, and 5-(N-methyl-N-isobutyl)-amiloride, respectively, were obtained from whole-vacuole recordings. The high inhibitory action (affinity) of amiloride and its analogs for the tonoplast cation channel suggests that these compounds could be used for the isolation and biochemical characterization of this protein.
Phosphorylation of polypeptides in membrane fractions from barley (Hordeum vulgare L. cv CM 72) roots was compared in in vitro and in vivo assays to assess the potential role of protein kinases in modification of membrane transport. Membrane fractions enriched in endoplasmic reticulum, tonoplast, and plasma membrane were isolated using sucrose gradients and the membrane polypeptides separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis. When the membrane fractions were incubated with γ-[32P]ATP, phosphorylation occurred almost exclusively in the plasma membrane fraction. Phosphorylation of a band at 38 kilodaltons increased as the concentration of Mg2+ was decreased from millimolar to micromolar levels. Phosphorylation of bands at 125, 86, 58, 46, and 28 kilodaltons required millimolar Mg2+ concentrations and was greatly enhanced by Ca2+. When roots of intact plants were labeled with [32P]orthophosphate, polypeptides at approximately 135, 116, 90, 46 to 53, 32, 28, and 19 kilodaltons were labeled in the plasma membrane fraction and polypeptides at approximately 73, 66, and 48 kilodaltons were labeled in the tonoplast fraction. Treatment of the roots of intact plants with 150 millimolar NaCl resulted in increased phosphorylation of some polypeptides while treatment with 100 mm NaCl had no effect.
Vacuoles are the stores for large amounts of stachyose [αgal (1,6) αgal (1,6) αglc (1,2) βfru] in tubers of Japanese artichoke (Stachys sieboldii). The uptake of stachyose by these vacuoles was examined and compared with that of sucrose. The uptake mechanisms of both sugars were quite similar. The kinetics showed a single saturable response to increasing external concentrations of 14C-sugars with similar apparent Km values of about 50 and 30 millimolar for stachyose and sucrose, respectively. The uptake rates, however, were always higher for stachyose than for sucrose. Stachyose and sucrose uptake was inhibited by fructose and raffinose, and, reciprocally, by sucrose and stachyose, but not by glucose or galactose. The main structural feature common to all sugars recognized by the uptake systems seems to be a terminal fructosyl residue. The uptake of both sugars was stimulated by Mg-ATP and inorganic pyrophosphate, suggesting a proton-sugar antiport system. The possibility that stachyose and sucrose might be transported by the same carrier is discussed.
The activation of spinach (Spinacia oleracea) chloroplast coupling factor 1 (CF1) by thioredoxin (ThR) was characterized using membrane-bound and soluble CF1. Light generates an electrochemical proton gradient across the thylakoid membrane, which increases the accessibility of the disulfide bond on the γ-subunit of CF1 to reduced ThR. The proton gradient substantially accelerates the activation of CF1 compared with thylakoids incubated in the dark with similar concentrations of dithiothreitol and ThR. The interaction of soluble CF1 with ThR was studied using fluorescent probes. CF1 in solution, with and without its associated ε-subunit, was labeled at Cys-322 of the γ-subunit with fluoresceinyl maleimide. ThR from Escherichia coli was labeled with eosin isothiocyanate. Labeled ThR and CF1 showed normal activities. Fluorescence energy transfer between donor fluoresceinyl maleimide and acceptor eosin isothiocyanate, manifested by a quenching of the donor fluorescence, was detected, suggesting that ThR and CF1 form an intermolecular complex. When the ε-subunit was absent, quenching of donor fluorescence was approximately doubled, indicating that labeled ThR could approach more closely to the γ-subunit of CF1. The distance between the fluorescent probes on CF1 and ThR was calculated to be approximately 65 Å when ε-subunit was present and 52 Å when ε was absent. These values are consistent with other distance measurements and energy transfer values reported previously for fluorescent probes on CF1. Whereas the extent of quenching increased by removal of the ε-subunit...
Reversible phosphorylation of proteins regulates numerous aspects of cell
function, and abnormal phosphorylation is causal in many diseases. Pyruvate
dehydrogenase complex (PDC) is central to the regulation of glucose
homeostasis. PDC exists in a dynamic equilibrium between de-phospho-(active)
and phosphorylated (inactive) forms controlled by pyruvate dehydrogenase
phosphatases (PDP1,2) and pyruvate dehydrogenase kinases (PDK1–4). In
contrast to the reciprocal regulation of the phospho-/de-phospho cycle of PDC
and at the level of expression of the isoforms of PDK and PDP regulated by
hormones and diet, there is scant evidence for regulatory factors acting
in vivo as reciprocal “on-off” switches. Here we show
that the putative insulin mediator inositol phosphoglycan P-type (IPG-P) has a
sigmoidal inhibitory action on PDK in addition to its known linear stimulation
of PDP. Thus, at critical levels of IPG-P, this sigmoidal/linear model
markedly enhances the switchover from the inactive to the active form of PDC,
a “push-pull” system that, combined with the developmental and
hormonal control of IPG-P, indicates their powerful regulatory function. The
release of IPGs from cell membranes by insulin is significant in relation to
diabetes. The chelation of IPGs with Mn2+ and Zn2+
suggests a role as “catalytic chelators” coordinating the traffic
of metal ions in cells. Synthetic inositol hexosamine analogues are shown here
to have a similar linear/sigmoidal reciprocal action on PDC exerting push-pull
Yea, Kyungmoo; Kim, Jaeyoon; Yoon, Jong Hyuk; Kwon, Taewan; Kim, Jong Hyun; Lee, Byoung Dae; Lee, Hae-Jeong; Lee, Seung Jae; Kim, Jong-In; Lee, Taehoon G.; Baek, Moon-Chang; Park, Ho Seon; Park, Kyong Soo; Ohba, Motoi; Suh, Pann-Ghill; Ryu, Sung Ho
Fonte: American Society for Biochemistry and Molecular BiologyPublicador: American Society for Biochemistry and Molecular Biology
Glucose homeostasis is maintained by the orchestration of peripheral glucose utilization and hepatic glucose production, mainly by insulin. In this study, we found by utilizing a combined parallel chromatography mass profiling approach that lysophosphatidylcholine (LPC) regulates glucose levels. LPC was found to stimulate glucose uptake in 3T3-L1 adipocytes dose- and time-dependently, and this activity was found to be sensitive to variations in acyl chain lengths and to polar head group types in LPC. Treatment with LPC resulted in a significant increase in the level of GLUT4 at the plasma membranes of 3T3-L1 adipocytes. Moreover, LPC did not affect IRS-1 and AKT2 phosphorylations, and LPC-induced glucose uptake was not influenced by pretreatment with the PI 3-kinase inhibitor LY294002. However, glucose uptake stimulation by LPC was abrogated both by rottlerin (a protein kinase Cδ inhibitor) and by the adenoviral expression of dominant negative protein kinase Cδ. In line with its determined cellular functions, LPC was found to lower blood glucose levels in normal mice. Furthermore, LPC improved blood glucose levels in mouse models of type 1 and 2 diabetes. These results suggest that an understanding of the mode of action of LPC may provide a new perspective of glucose homeostasis.