Charge and acidity compensation during proton-sugar symport in Chlorella: The H+-ATPase does not fully compensate for the sugar-coupled proton influx

This study was undertaken in order to demonstrate the extent to which the activity of the plasmalemma H⁺-ATPase compensates for the charge and acidity flow caused by the sugar-proton symport in cells of chlorella vulgaris Beij.. Detailed analysis of H⁺ and K⁺ fluxes from and into the medium together with measurements of respiration, cytoplasmic pH, and cellular ATP-levels indicate three consecutive phases after the onset of H⁺ symport. Phase 1 occurred immediately after addition of sugar, with an uptake of H⁺ by the hexoseproton symport and charge compensation by K⁺ loss from the cells and, to a smaller degree, by loss of another ion, probably a divalent cation. This phase coincided with strong membrane depolarization. Phase 2 started approximately 5 s after addition of sugar, when the acceleration of the H⁺-ATPase caused a slow-down of the K⁺ efflux, a decrease in the cellular ATP level and an increase in respiration. The increased respiration was most probably responsible for a pronounced net acidification of the medium. This phase was inhibited in deuterium oxide. In phase 3, finally, a slow rate of net H⁺ uptake and K⁺ loss was established for several further minutes, together with a slight depolarization of the membrane. There was hardly any pH change in the cytoplasm, because the cytoplasmic buffering capacity was high enough to stabilize the pH for several minutes despite the net H⁺ fluxes. The quantitative participation of the several phases of H⁺ and K⁺ flow depended on the pH of the medium, the ambient Ca²⁺ concentration, and the metabolic fate of the transported sugar. The results indicate that the activity of the H⁺-ATPase never fully compensated for H⁺ uptake by the sugar-symport system, because at least 10% of symport-caused charge inflow was compensated for by K⁺ efflux. The restoration of pH in the cytoplasm and in the medium was probably achieved by metabolic reactions connected to increased glycolysis and respiration.Abbreviations DMO dimethyloxazolidinedione - EDTA ethylcnediaminetetraacetic acid - p.c. packed cell volume     

The link between the electrogenic and redox functions of the plasmalemma and the energy metabolism of the cell

A dependence of the plasmalemma redox activity, determined by the reduction of external electron acceptors (ferricyanide, nitro-blue tetrazolium), on the energy state of the cell, which was modified by light conditions or introduction of glucose into the media, was shown on leaves of Elodea canadensis Rich. Glucose (10 m M ) and light (40 W m^-2) caused hyperpolarization of the membrane potential and stimulated the redox activity of the plasmalemma. 3-(3,4-Dichlorophenyl)-1,1-dimethylurea (DCMU) completely inhibited the light activation of electrogenic and redox functions of the plasmalemma. The light saturation intensity for membrane potential and ferricyanide reductase activity was 10–30% of the light saturation of photosynthesis. Membrane potential, K⁺ transport and plasmalemma redox activity changed in parallel in response to light and darkness and when DCMU was added. Ferricyanide reductase activity is suggested to be a simple parameter for characterizing the energy state of the cell. The functional significance of the light-induced hyperpolarization of the membrane potential is discussed.     

Identification of a calmodulin-stimulated (Ca2++ Mg2+)-ATPase in a plasma membrane fraction isolated from maize (Zea mays) leaves

Plasma membranes were isolated from light-grown, 14-day-old maize leaves ( Zea mays L . cv. Golden Cross Bantam) using aqueous two-phase partitioning. The plasma membrane (PM) fraction contained < 0.3% of the total chlorophyll, < 0.2% of the mitochondrial marker enzyme activity, minimal contamination by endomembranes and 34% of the total PM.
A calmodulin-stimulated (Ca2++ Mg2+)-ATPase was identified in the PM-enriched fraction. The Ca2++ calmodulin stimulation was dependent on Mg2+, saturated at ca 25 μM total Ca2+, had a pH maximum at 7.2 and was maximally stimulated by 600 n M bovine brain calmodulin. The stimulation was not greatly affected by the anion present and showed a divalent cation specificity of Ca2+ > Sr+2 ± Mn+2 > Co2+± Cu2+ > Ba2+. The napthalenesulfonamide W7, an antagonist of calmodulin action, completely inhibited the calmodulin stimulation at 175 μM , while the less active analogue W5 was ineffective at this concentration. La3+, an inhibitor of PM Ca2+ transport, showed a 50% inhibition of calmodulin-stimulated ATPase activity at ca 200 μM . Taken as a whole, these data demonstrate the presence of a calmodulinstimulated, (Ca2++ Mg2+)-ATPase on the cytoplasmic surface of the plasma membrane of maize leaf cells.     

Changes in parameters of the plasmalemma ATPase during cold acclimation of apple (Mains domestica) tree bark tissues

A plasmalemma fraction was isolated from homogenized apple tree (Mains domestica Borkh 'Golden Delicious') hark tissues using aqueous phase partitioning and ultra-centrifugation. Results of marker enzyme assays indicated that a membrane preparation highly enriched in plasma membranes was obtained. ATPase activity in this preparation possessed a high specificity for ATP as substrate, was inhibited by vanadate, diethylstilbcsterol and dicyclohexylcarbocHimide, and was insensitive to inhibitors of mitochondria! and tonoplasl ATPases. Specific activity of the plasma-lemma ATPase increased during cold acclimation prior to the attainment of vegetative maturity. Kinetic parameters (Km, Vln) determined from assays performed at different temperatures (10, 30°C) indicated a differential effect of cold acclimation on enzyme activity. Vm increased during cold acclimation, whereas Km increased when determined at 30°C but declined at 10°C. Acclimation treatments during April and May resulted in alteration of ATPase kinetics in the absence of any increase in bark frost hardiness. Changes in ATPase kinetics may be related more to enhanced low temperature metabolism than to frost hardiness per se.     

Effects of gamma irradiation on the plasma membrane of suspension-cultured apple cells. Rapid irreversible inhibition of H+-ATPase activity

The effects of ionizing radiation, used in post-harvest treatment of fruit and vegetables. were investigated on cultured apple cells ( Pyrus malus L. cv. Royal Red) on a short-term period. Irradiation (2 kGy) induced an increase of passive ion effluxes from cells and a decrease of cell capacity to regulate external pH. These alterations are likely due to effects on plasma membrane structure and function and were further investigated by studying the effects of irradiation on plasma membrane H⁺-ATPase activity. Plasma membrane-enriched vesicles were prepared and the H⁺-ATPase activity was characterized. Irradiation of the vesicles induced a dose dependent inhibition of H⁺-ATPase activity. The loss of enzyme activity was immediate, even at low doses (0.5 kGy), and was not reversed by the addition of 2m M dithiothreitol. This inhibition may be the result of an irreversible oxidation of enzyme sulfhydryl moieties and/or the result of changes induced within the lipid bilayer affecting the membrane-enzyme interactions. Further analysis of the H⁺-ATPase activity was carried out on vesicles obtained from irradiated cells confirming the previous results. In vivo recovery of activity was not observed within 5 h following the treatment, thus explaining the decrease of cell capacity to regulate external pH.
This rapid irreversible inhibition of the plasma membrane H⁺-ATPase must be considered as one of the most important primary biochemical events occurring in irradiated plant material.     

In vivo binding of auxin to the plasmalemma and tonoplast of parenchymal cells in the wheat coleoptile

Tritiated auxin applied by an agar block on the wheat coleoptile tip for 2 hr was covalently fixed to adjacent protein by treatment with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (DCC). The density of labelled auxin in the nucleus, the cell wall, the cytoplasm, and the vacuole was determined by autoradiography. Localization of tritiated auxin was studied at high resolution at the tonoplast and the plasmalemma lining the transverse (distal and proximal) and the longitudinal walls. The radioactivity along the tonoplast was always less than along the plasma membrane. The distribution of ³H-auxin was different across the longitudinal and transverse regions of the plasmalemma. The labelling was distributed asymmetrically on the longitudinal plasma membrane with a peak observed on the external surface. Tritiated auxin was distributed more symmetrically on the distal and the proximal plasma membranes. Our results are in agreement with the hypothesis that there are 2 different specific binding sites on the plasmalemma. The ratio of auxin present at the proximal and distal regions of the plasmalemma was 1.28.     

Very long chain fatty acids: Occurrence and biosynthesis in membrane fractions from etiolated maize coleoptiles

The endoplasmic reticulum from maize coleoptiles elongates stearoyl-CoA more effectively than the plasmalemma-enriched fraction. The alkane and very lo     

Maltose uptake in the Zea mays scutellum

Maltose transport in slices of the maize scutellum was demonstrated despite the presence of an active maltase situated at the cell surface. The maltase could be inhibited or destroyed by treatments (neutral pH during uptake, pretreatment in Tris buffer at pH 7·5, or in 0·01 N HCl) that allowed appreciable rates of maltose uptake to occur. Using Tris- and HCl-treated slices, it was found that at disaccharide concentrations of 50 and 100 mM, maltose and sucrose were taken up at very nearly the same rates. At sugar concentrations below 50 mM, sucrose was taken up at greater rates than maltose. The maltose content of the slices was directly proportional to the maltose concentration of the bathing solution, and about 4 hr were required for equilibration. From this, it is concluded that one way maltose enters the slices is by free or facilitated diffusion. However, endogenous maltose is utilized by the slices at rates that are much too low to account for the net rates of maltose uptake. Although the slices contain a high level of surface maltase activity, only a low level of endogenous maltase activity was found. This probably accounts for the slow utilization of endogenous maltose. Therefore, the existence of a specific maltose transport system is proposed; a system that contains a carrier saturable with maltose, but one that does not release free maltose into the cytoplasm.     

Assembly and plasma membrane targeting of recombinant immunoglobulin chains in plants with a murine immunoglobulin transmembrane sequence

The cDNA encoding a full-length murine immunoglobulin 1 heavy chain with its native leader sequence, transmembrane and intracellular domains was introduced into transgenic plants. Transformed plants expressed the recombinant polypeptide, but, in contrast to plants expressing the heavy chain without transmembrane sequence, the protein appeared to be associated with a plant cell membrane. Extraction of the membrane-associated heavy chain required the presence of a non-ionic detergent, and immunofluorescence studies of protoplasts demonstrated surface expression of membrane Ig heavy chain on up to 40% of the cells from a transgenic leaf. In plants expressing both the membrane Ig heavy chain and its partner light chain, functional antibody was also localised to the plant cell membrane and retention of the heavy chain at this site appeared to have no effect on the efficiency of antibody assembly. This approach of localising and accumulating recombinant antibody in cell membranes may have a number of applications, including passive immunisation against plant pathogens.     

Anomalous kinetics of sucrose uptake in maize scutellum slices1

Abstract The kinetics of sucrose uptake into maize scutellum slices showed that the uptake mechanism had a saturable component with a Km of l.5mol m^?3 sucrose. Nevertheless, uptake rate was constant (zero order) over extended periods of time until the bathing solution was nearly depleted of sucrose. It is concluded that these anomalous uptake kinetics reflect sucrose influx across the plasmalemma because of the following results: (a) Efflux of sucrose into buffer was negligible compared with uptake rate, (b) When slices were incubated in fructose, sucrose was synthesized and there was a net release of sucrose to the bathing solution until a steady-state was reached when influx and efflux were equal in magnitude. After the steady-state was reached, efflux of sucrose from the slices was nearly the same in magnitude as the estimated rate of uptake that would have occurred from bathing solutions initially containing the steady-state sucrose concentration, (c) Exchange of sucrose between bathing solution and slices was negligible compared with uptake rate, (d) Pretreatment of slices with uranyl nitrate abolished sucrose uptake, but uptake rate was re-established in these slices after treatment with HCl (pH 2). Uptake rate was set by the initial sucrose concentration of the bathing solution, and was not influenced by the level of endogenous sucrose or by the rate at which the sucrose concentration of the bathing solution declined. Abrupt increases in sucrose concentration during the uptake period increased the rate of uptake only if the concentration was increased above that at the start of the uptake period. Following abrupt decreases in sucrose concentration, there was a lag of about 30 min before uptake rate decreased greatly. If slices were washed and replaced in a fresh sucrose solution during the uptake period, a new uptake rate was set to correspond to the new initial sucrose concentration. It is suggested that the sucrose carrier has a transport site with a relatively low Km (much below 1.5mol m^?3) and that the measured Km (1.5mol m^?3) is that of a site that binds sucrose and thereby controls the rate of uptake. The low Km suggested for the transport site would explain the zero order kinetics but a model of the uptake mechanism that includes the control site cannot, as yet, be constructed from the data.