A tonoplast enriched fraction was obtained from Zea mays L. coleoptiles by isopycnic centrifugation of microsomal membranes in a sucrose step gradient. At the 18/26% interface chloride-stimulated and nitrate-inhibited proton pumping activity coincided with a Mg2+-ATP dependent accumulation of 3-O-methyl-d-glucose (OMG) as determined by a membrane filtration technique using 14C-labeled substrate. OMG transport showed an apparently saturable component with a Km of 110 micromolar, and was completely inhibited by 10 micromolar carbonyl cyanide m-chlorophenylhydrazone. Polyclonal antibodies against solubilized native tonoplast H+-ATPase and its 62 and 72 kilodalton subunits were assayed for their ability to inhibit proton pumping and OMG accumulation. Antibodies against both the native enzyme and the putative catalytic subunit (72 kilodalton) strongly inhibited proton pumping and OMG transport whereas antibodies against the 62 kilodalton subunit had only a slight effect on both processes.
Acridine orange altered the response to anions of both ATP and in-organic pyrophosphate-dependent pH gradient formation in tonoplast vesicles isolated from oat (Avena sativa L.) roots and red beet (Beta vulgaris L.) storage tissue. When used as a fluorescent pH probe in the presence of I−, ClO3−, NO3−, Br−, or SCN−, acridine orange reported lower pH gradients than either quinacrine or [14C]methylamine. Acridine orange, but not quinacrine, reduced [14C]methylamine accumulation when NO3− was present indicating that the effect was due to a real decrease in the size of the pH gradient, not a misreporting of the gradient by acridine orange. Other experiments indicated that acridine orange and NO3− increased the rate of pH gradient collapse both in tonoplast vesicles and in liposomes of phosphatidylcholine and that the effect in tonoplast vesicles was greater at 24°C than at 12°C. It is suggested that acridine orange and certain anions increase the permeability of membranes to H+, possibly because protonated acridine orange and the anions form a lipophilic ion pair within the vesicle which diffuses across the membrane thus discharging the pH gradient. The results are discussed in relation to the use of acridine orange as a pH probe. It is concluded that the recently published evidence for a NO3−/H+ symport involved in the export of NO3− from the vacuole is probably an artefact caused by acridine orange.
An ATP-dependent protein kinase was partially purified from isolated outer envelope membranes of pea (Pisum sativum L., Progress No. 9) chloroplasts. The purified kinase had a molecular weight of 70 kilodaltons, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was of the cyclic nucleotide and Ca2+, calmodulin-independent type. The purification involved the detergent solubilization of purified outer envelopes by 0.5% cholate and 1% octylglycoside, followed by centrifugation on a linear 6 to 25% sucrose gradient. Active enzyme fractions were further purified by affinity chromatography on histone III-S Sepharose 4B and ion exchange chromatography on diethylaminoethyl cellulose. The protein kinase eluted at 100 millimolar and 50 millimolar NaCl, respectively. The protein kinase was essentially pure as judged by Western blot analysis. The enzyme has a KM of 450 micromolar for ATP and a Vmax of 25 picomoles of 32P incorporated into histone III-S per minute per microgram. Inhibition by ADP is competitive (Ki 150 micromolar).
Chloride or nitrate decreased a pH gradient (measured as [14C]methylamine accumulation) in tonoplast-enriched vesicles. The ΔpH decrease was dependent on the anion concentration. These effects are independent of the anion-sensitive H+-ATPase of the tonoplast, since the pH gradient (acid inside) was imposed artificially using a pH jump or a K+ gradient and nigericin. 4,4′-Diisothiocyano-2,2′-stilbene disulfonic acid partially prevented the decrease in pH gradient induced by Cl−. Two possible models to account for this anion-dependent decrease of ΔpH are: (a) H+ loss is accompanied by Cl− or NO3− efflux from the vesicles via H+/anion symport systems on the tonoplast and (b) H+ loss is accompanied by Cl− or NO3− uptake into the vesicles via H+/anion antiport systems. Depending on the requirements and conditions of the cell, these two systems would serve to either mobilize Cl− and NO3− stored in the vacuole for use in the cytoplasm or to drive anions into the vacuole. Chloride or nitrate also decreased a pH gradient in fractions containing plasma membrane and Golgi, implying that these membranes may have similar H+-coupled anion transport systems.
Addition of ferredoxin to isolated thylakoid membranes reconstitutes electron transport from water to NADP and to O2 (the Mehler reaction). This electron flow is coupled to ATP synthesis, and both cyclic and noncyclic electron transport drive photophosphorylation. Under conditions where the NADPH/NADP+ ratio is varied, the amount of ATP synthesis due to cyclic activity is also varied, as is the amount of cyclic activity which is sensitive to antimycin A. Partial inhibition of photosystem II activity with DCMU (which affects reduction of electron carriers of the interphotosystem chain) also affects the level of cyclic activity. The results of these experiments indicate that two modes of cyclic electron transfer activity, which differ in their antimycin A sensitivity, can operate in the thylakoid membrane. Regulation of these activities can occur at the level of ferredoxin and is governed by the NADPH/NADP ratio.
Adenosine and guanosine are transported into Petunia hybrida pollen by a saturable, carrier-mediated mechanism. The energy poisons carbonylcyanide-m-chlorophenylhydrazone, 2,4-dinitrophenol, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, and N,N′-dicyclohexylcarbodiimide all inhibit uptake, suggesting an energy coupled (active) transport process. Transport takes place against a concentration gradient, strongly favoring an active transport mechanism. The purine nucleoside transport in Petunia pollen differs from that already reported for pyrimidine nucleosides in that it exhibits a significantly higher Km for nucleoside and is not so severely inhibited by the polyamine, spermine. Like that for the pyrimidine nucleosides uridine and cytosine, however, the system exhibits a broad pH optimum, is inhibited by sulfydryl-binding reagents, while the potent inhibitors of nucleoside transport in animal cells, nitrobenzylthioinosine and dipyridamole, have no effect. Transport of both purine and pyrimidine nucleosides in germinating pollen decreases steadily with time, a finding consistent with reports that RNA synthesis and DNA repair are early events of pollen germination and tube elongation. However, since these precursors are often used to demonstrate nucleic acid synthesis...
An anion-sensitive H+-translocating ATPase was identified in membrane vesicles isolated from mature green tomato (Lycopersicon esculentum) fruit. The H+-ATPase was associated with a low density membrane population having a peak density of 1.11 grams per cubic centimeter, and its activity was inhibited by NO3−, N,N′-dicyclohexylcarbodiimide and diethylstilbestrol but not by vanadate, azide, molybdate, or oligomycin. This H+-ATPase has an unusual pH dependence indicating both a slightly acidic and a near neutral peak of activity. Chloride was found to be a potent stimulator of ATPase activity. The Km for the H+-ATPase was approximately 0.8 millimolar ATP. The characteristics of this H+-ATPase are very similar to those described for a number of plant cell tonoplast H+-ATPases suggesting that the activity identified in tomato fruit membranes is tonoplast-associated. This report demonstrates the feasibility of isolating tonoplast vesicles from acidic fruit tissues for studies of transport activities associated with fruit development and maturation.
The characteristics of fusicoccin binding were investigated in microsomes from 24-h-old radish (Raphanus sativus L.) seedlings. The time course of fusicoccin binding depended on fusicoccin concentration: equilibrium was reached much faster at 10 nanomolar fusicoccin than at 0.3 nanomolar fusicoccin. Scatchard analysis of equilibrium binding as a function of fusicoccin concentration indicated a single class of receptor sites with a Kd of 1.8 nanomolar and a site density of 6.3 picomoles per milligram protein. Similar values (Kd 1.7 nanomolar and site density 7 picomoles per milligram protein) were obtained from the analysis of the dependence of equilibrium binding on membrane concentration at fixed fusicoccin concentrations. Fusicoccin binding comigrated with the plasma membrane H+-ATPase in an equilibrium sucrose density gradient: both activities formed a sharp peak (1.18 grams per milliliter) clearly distinct from that of markers of other membranes which all peaked at lower densities. The saturation profiles of fusicoccin binding and of fusicoccin-induced activation of the plasma membrane H+-ATPase, measured under identical conditions, were similar, supporting the view that fusicoccin-induced activation of the plasma membrane H+-ATPase is mediated by fusicoccin binding to its plasma membrane receptor.
The tonoplast ATPase from etiolated seedlings of Vigna radiata L. (mung bean) was isolated using a two-step detergent solubilization modified from Mandala and Taiz (S Mandala, L Taiz  Plant Physiol 78: 327-333). After ultracentrifugation on 10 to 28% sucrose gradient, the ATPase showed a 31.6-fold purification over the initial specific activity of the starting tonoplast-enriched membranes. The purified ATPase used Mg2+-ATP as the preferred substrate. The tonoplast ATPase was isolated in a form with characteristics similar to that on its native membrane environment. Analysis by SDS-PAGE revealed two prominent bands with molecular weights of 78,000 (α subunit) and 64,000 (β subunit). The intensity of Coomassie blue staining showed a 1:1 stoichiometry for α and β subunits. The amino acid composition of α and β subunits also confirmed the suggested stoichiometry of the subunit composition of the tonoplast ATPase. Moreover, radiation inactivation analysis yielded a functional size of 414 ± 24 and 405 ± 25 kilodaltons for soluble and membrane bound tonoplast ATPases, respectively. It is possible that the functioning tonoplast ATPase may be in a form of αβ-heteromultimer.
Maize (Zea mays L.) caryopses were grown in the presence of fenpropimorph, a systemic fungicide, for 7 days in the dark. Membrane fractions enriched, respectively, in endoplasmic reticulum, plasma membrane, and mitochondria were isolated from control and treated maize roots and analyzed for their free sterol, phospholipid, and fatty acid composition. In treated plants, the intracellular distribution of free sterols was dramatically modified both qualitatively and quantitatively. The normally occurring Δ5-sterols disappeared almost completely and were replaced by 9β, 19-cyclopropyl sterols, mainly cycloeucalenol and 24-methyl pollinastanol. These new compounds were found to accumulate in all the membrane fractions in such a way that the endoplasmic reticulum-rich fraction became the richest one in free sterols instead of the plasma membrane. In contrast, the fenpropimorph treatment of maize roots was shown not to affect either the relative proportions or the amounts of the individual phospholipids, but an increase in the unsaturation index of phospholipid-fatty acyl chains of the endoplasmic reticulum-rich fraction was observed. The present data suggest that, in higher plant membranes, cyclopropyl sterols could play a structural role similar to that of the bulk of Δ5-sterols.
Intact chloroplasts were compared to isolated thylakoids as to whether storage of the organelle in high KCl medium caused the energy coupling reactions to show a delocalized or a localized proton gradient energy coupling response. With isolated thylakoids, the occurrence of one or the other energy coupling mode can be reversibly controlled by the concentration of mono- and divalent cations used for the thylakoid storage media. Calcium was shown to be the key ion and previous evidence suggested a Ca2+-controlled gating of H+ fluxes in the thylakoid membrane system (G Chiang, RA Dilley  Biochemistry 26: 4911-4916). Isolated, intact chloroplasts, which retained the outer envelope membranes during the 30 min or longer storage treatments in various concentrations of KCl and CaCl2 (with sorbitol to maintain iso-osmotic conditions), were osmotically burst in a reaction cuvette and within 3 minutes were assayed for either a localized or a delocalized proton gradient energy coupling (ATP formation) mode. The intact chloroplast system was analogous to isolated thylakoids, with regard to the effects of KCl and CaCl2 on the energy coupling mode. For example, adding 100 millimolar KCl to the intact organelle storage medium resulted in the subsequent ATP formation assay showing delocalized proton gradient coupling just as with isolated thylakoids. Adding 5 millimolar CaCl2 to the 100 millimolar KCl storage medium resulted in a localized proton gradient coupling mode. Suspending thylakoids in stromal material previously isolated from intact chloroplast preparations and testing the energy coupling response showed that the stromal milieu has enough Ca2+ to cause the localized coupling response even though there was about 80 millimolar K+ in the intact chloroplasts used in this study (determined by atomic absorption spectrophotometry). Extrapolating the intact chloroplast data to the whole leaf level...
Five- and six-subunit forms of F1-ATPase were purified from pea (Pisum sativum L. cv Homesteader) cotyledon submitochondrial particles. Apart from the usual complement of five subunits, the six-subunit enzyme contained an additional 26,500-dalton protein. Both forms of the F1-ATPase were used to reconstitute oxidative phosphorylation in F1-depleted (ASU) as well as in F1 and oligomycin-sensitivity conferring protein (OSCP)-depleted (ASUA) bovine mitochondrial membranes. The six-subunit enzyme was considerably more efficient in reconstituting the ATP synthesis than the five-subunit enzyme. Both forms of the enzyme were also able to reconstitute the ATPase activity in ASU- as well as in ASUA-particles. There were substantial differences, however, in the oligomycin sensitivity of the ATPase bound to the ASUA-particles: 20 and 60% inhibition by oligomycin was obtained in the case of the five-subunit and six-subunit enzyme, respectively. We conclude, that the 26,500-dalton protein present in the six-subunit F1-ATPase is responsible for the increase in oligomycin sensitivity of the bound enzyme and functions, therefore, as the plant OSCP.
The phosphate translocator was identified in the envelope membranes of both mesophyll and bundle sheath chloroplasts of Panicum miliaceum L. by labeling with [1,2-3H]1,2-(2,2′ -disulfo-4,4′ -diisothiocyano)diphenylethane ([3H]H2DIDS) and by using SDS-PAGE. Assay of 32Pi uptake by the chloroplasts showed that the phosphate translocators of both types of chloroplasts have a higher affinity for phosphoenolpyruvate than the C3 counterpart and can be regarded as C4 types.
Plastids were isolated from a plastome mutator-induced mutant (pm7) of Oenothera hookeri and were analyzed for various physiological and biochemical attributes. No photosynthetic electron transport activity was detected in the mutant plastids. This is consistent with previous ultrastructural analysis showing the absence of thylakoid membranes in the pm7 plastids and with the observation of aberrant processing and accumulation of chloroplast proteins in the mutant. In comparison to wild type, the mutant tissue lacks chlorophyll, and has significant differences in levels of four fatty acids. The analyses did not reveal any differences in carotenoid levels nor in the synthesis of several chloroplast lipids. The consequences of the altered composition of the chloroplast membrane are discussed in terms of their relation to the aberrant protein processing of the pm7 plastids. The pigment, fatty acid, and lipid measurements were also performed on two distinct nuclear genotypes (A/A and A/C) which differ in their compatibility with the plastid genome (type I) contained in these lines. In these cases, only chlorophyll concentrations differed significantly.
Barley (Hordeum vulgare L. cv Halcyon) seedlings which had been grown in full strength complete inorganic nutrient media (containing 6 millimolar K+) had high internal K+ concentrations and low values of K+ (86Rb+) influx when influx was measured from solutions containing 100 micromolar K+. Transfer of these plants to solutions lacking K+ resulted in significant reductions of root and shoot K+ concentrations and values of K+ (86Rb+) influx increased by greater than 10-fold within 3 days. When plants treated in this way were returned to complete solutions, containing K+, the changes induced by K+ deprivation were reversed. Parallel studies of microsomal membranes by means of SDS-PAGE demonstrated that the expression of a group of polypeptides increased or decreased in parallel with changes of K+ (86Rb+) influx. Most prominent of these were 45 and 34 kilodalton polypeptides which specifically responded to K+ status of the barley plants; their expression was not enhanced by N or P deprivation. The 45 kilodalton polypeptide was susceptible to degradation by a membrane associated protease when microsomes were washed in buffer containing 0.2 millimolar PMSF. This loss was prevented by increasing PMSF concentration to 2 millimolar.
The ferri-reductase activity of whole cells of Saccharomyces cerevisiae (washed free from the growth medium) was markedly increased 3 to 6 h after transferring the cells from a complete growth medium (preculture) to an iron-deficient growth medium (culture). This increase was prevented by the presence of iron, copper, excess oxygen, or other oxidative agents in the culture medium. The cells with increased ferri-reductase activity had a higher reduced glutathione content and a higher capacity to expose exofacial sulfhydryl groups. Plasma membranes purified from those cells exhibited a higher reduced nicotinamide adenine phosphate (NADPH)-dependent ferri-reductase specific activity. However, the intracellular levels of NADPH, NADH, and certain organic acids of the tricarboxylic acids cycle were unchanged, and the activity of NADPH-generating enzymes was not increased. Addition of Fe(III)-EDTA to iron-deprived and iron-rich cells in resting suspension resulted in a decrease in intracellular reduced glutathione in the case of iron-deprived cells and in an increase in organic acids and a sudden oxidation of NADH in both types of cells. The depolarizing effect of Fe3+ was more pronounced in iron-rich cells. The metabolic pathways that may be involved in regulating the trans-plasma membrane electron transfer in yeast are discussed.
Phosphatidylinositol kinase (PI), phosphatidylinositol monophosphate (PIP) kinase, and diacylglycerol (DAG) kinase activities were detected in the cytoskeletal fraction isolated from microsomes and plasma membranes of carrot (Daucus carota L.) cells grown in suspension culture. The lipid kinase activities were associated with the actin filament fraction (F-actin fraction) isolated from the cytoskeleton. The PI and PIP kinase activity in the F-actin fraction significantly increased after cells were treated with Driselase, a mixture of cell wall-degrading enzymes; however, the DAG kinase activity in the F-actin fraction was unaffected by the Driselase treatment. These data indicate that at least one form of PI, PIP, and DAG kinase preferentially associates with actin filaments and/or actin binding proteins and that cytoskeletal-associated PI and PIP kinase activities can change in response to external stimulation.
Effective ionophore:chlorophyll ratios were determined for various ionophores that decrease the electrical potential across thylakoid membranes in intact and hypo-osmotically lysed chloroplasts isolated from spinach (Spinacia oleracea). The efficacy of gramicidin D, valinomycin, carbonylcyanide m-chlorophenylhydrazone, and dicyclohexano-18-crown-6 in collapsing the electrical potential was determined spectrophotometrically by the decay half-time of the absorbance change at 518 nanometers induced by a saturating, single turnover flash. The results show that the effectiveness of the ionophores in collapsing the electrical potential in intact and lysed chloroplasts depends on the amount of ionophore-accessible membrane in the assay medium. Only gramicidin exhibited a significant difference in efficacy between intact and lysed chloroplasts. The ratio of gramicidin to chlorophyll required to collapse the electrical potential was more than 50 times higher in intact chloroplasts than in lysed chloroplasts. The efficacy of carbonylcyanide m-chlorophenylhydrazone was significantly reduced in the presence of bovine serum albumin. The other ionophores tested maintained their potency in the presence of bovine serum albumin. Valinomycin was the most effective ionophore tested for collapsing the electrical potential in intact chloroplasts...
The phosphorylation of thylakoid membranes in the Chromophyte alga Ochromonas danica was studied in whole cells and in vitro. Protein kinase activity was observed in the thylakoid fraction, and several membrane-bound polypeptides were found to be phosphorylated. The thylakoid protein kinase demonstrated several unusual regulatory properties. Both the polypeptides that were phosphorylated and the rate of protein phosphorylation were independent of illumination. Protein kinase activity was also unaffected by 3-(3,4-dichlorophenyl)-1,1-dimethylurea, diuron. The kinase activity was inhibited under strong reducing conditions. Whole cells labeled with 32PO43− were converted to light states I and II by pre-illumination favoring photosystem I or photosystem II, respectively. Analysis of the phosphoproteins from cells in state I and state II showed that no changes in phosphorylation accompanied the change in energy redistribution.
In comparison with other cell organelles, the Dunaliella salina plasma membrane was found to be highly enriched in phospholipase C activity toward exogenous [3H]phosphatidylinositol 4,5-bisphosphate (PIP2). Based on release of [3H]inositol phosphates, the plasma membrane exhibited a PIP2-phospholipase C activity nearly tenfold higher than the nonplasmalemmal, nonchloroplast `bottom phase' (BP) membrane fraction and 47 times higher than the chloroplast membrane fraction. The majority of phospholipase activity was clearly of a phospholipase C nature since over 80% of [3H]inositol phosphates released were recovered as [3H]inositol trisphosphate (IP3). These results suggest a plausible mechanism for the rapid breakdown of PIP2 and phosphatidylinositol 4-phosphate (PIP) following hypoosmotic shock. Quantitative analysis of major [3H]inositol phospholipids during these assays revealed that some of the [3H]-PIP2 was converted to [3H]phosphatidylinositol 4-monophosphate (PIP) and to [3H]phosphatidyl-inositol (PI) in the BP fraction of membrane remaining after removal of plasmalemma and chloroplasts. This latter fraction is enriched more than fivefold in PIP2/PIP phosphomonoesterase activity when compared to the plasmalemma or chloroplast membrane fractions. We have also examined some of the in vitro characteristics of the plasma membrane phospholipase C activity and have found it to be calcium sensitive...