Página 3 dos resultados de 686 itens digitais encontrados em 0.011 segundos

Polyphosphoinositides Are Present in Plasma Membranes Isolated from Fusogenic Carrot Cells 1

Wheeler, Jeffery J.; Boss, Wendy F.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /10/1987 EN
Relevância na Pesquisa
578.8073%
Fusogenic carrot cells grown in suspension culture were labeled 12 hours with myo-[2-3H]inositol. Plasma membranes were isolated from the prelabeled fusogenic carrot cells by both aqueous polymer two-phase partitioning and Renografin density gradients. With both methods, the plasma membrane-enriched fractions, as identified by marker enzymes, were enriched in [3H]inositol-labeled phosphatidylinositol monophosphate (PIP) and phosphatidylinositol bisphosphate (PIP2). An additional [3H]inositol-labeled lipid, lysophosphatidylinositol monophosphate, which migrated between PIP and PIP2 on thin layer plates, was found primarily in the plasma membrane-rich fraction of the fusogenic cells. This was in contrast to lysophosphatidylinositol which is found primarily in the lower phase, microsomal/mitochrondrial-rich fraction.

separation and Immunological Characterization of Membrane Fractions from Barley Roots 1

DuPont, Frances M.; Tanaka, Charlene K.; Hurkman, William J.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /03/1988 EN
Relevância na Pesquisa
574.88438%
Tonoplast and plasma membranes (PM) were isolated from barley roots (Hordeum vulgare L. cv California Mariout 72) using sucrose step gradients. The isolation procedure yielded sufficient quantities of PM and tonoplast vesicles that were sealed and of the right orientation to measure ATP-dependent proton transport in vitro. The proteins of the endoplasmic reticulum, tonoplast-plus-Golgi membrane (TG) and PM fractions were separated on sodium dodecyl sulfate gels, and immunoblots were used to test for cross-contamination between the fractions. Proteins that cross-reacted with antibodies to the PM ATPase from corn roots and Neurospora were greatly enriched in the PM fraction, as were proteins that cross-reacted with monoclonal antibodies to an arabinogalactan protein from the PM of tobacco cells. Proteins that cross-reacted with antibodies to the 58- and 72-kilodalton subunits of the tonoplast ATPase of red beet storage tissue were greatly enriched in the TG fraction. The results with immunoblots and enzyme assays indicated that there was little cross-contamination between the tonoplast and PM vesicles. The molecular weights and isoelectric points of the PM ATPase and the tonoplast ATPase subunits were also determined using immunoblots of two-dimensional gels of the PM and TG proteins.

Characterization of the H+ Translocating Adenosine Triphosphatase and Pyrophosphatase of Vacuolar Membranes Isolated by Means of a Perfusion Technique from Chara corallina1

Takeshige, Kazuhiko; Tazawa, Masashi; Hager, Achim
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /04/1988 EN
Relevância na Pesquisa
574.78176%
Sealed tonoplast vesicles were isolated from single cells of Chara corallina with the aid of an intracellular perfusion technique in combination with a 3/10% Percoll two step gradient centrifugation. The isolated tonoplast fraction was free from plasmalemma and chloroplasts, and showed no activities of cytochrome c oxidase, and latent IDPase, but had about 10% of the NADH-cytochrome c reductase activity. The vesicles had both ATPase and PPase activities, which could be stimulated in the presence of 10 micromolar gramicidin by 170 and 130%, respectively, demonstrating the existence of sealed vesicles. Furthermore, ATP- and PPi-dependent H+ pumping through the membrane into the vesicles was shown. Both ATPase and PPase had pH optima around pH 8.5. At the physiological pH, 7.3, they still had more than 80% of their maximal activities. Ammonium molybdate, azide, and vanadate had no or little effect on the activities of both enzymes or their associated H+ pumping activities. N,N′-dicyclohexylcarbodiimide inhibited the ATPase strongly (I50 = 20 micromolar) but the PPase only weakly. The ATPase was also more sensitive to N-ethylmaleimide than the PPase. 4,4′-Stilbenedisulfonic acid affected both enzyme activities and their associated H+ pumping activities. This is in contrast to the H+-PPase of higher plants which is 4...

Plant Plasma Membrane Proteins 1: II. Biotinylation of Daucus Carota Protoplasts and Detection of Plasma Membrane Polypeptides after Sds-Page

Grimes, Howard D.; Slay, Raymond M.; Hodges, Thomas K.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /10/1988 EN
Relevância na Pesquisa
574.82113%
The ability of two biotinylating reagents, sulfosuccinimidobiotin and sulfosuccinimidyl 2-(biotinamido)ethyl-1,3′-dithiopropionate, to label plasma membrane proteins was examined. These compounds form covalent bonds with the free amino groups of proteins and label the proteins with biotin. Biotinylated proteins can be detected with avidin-peroxidase staining. Protoplasts isolated from embryogenic Daucus carota suspension cells were labeled with biotin and the membranes were separated on linear sucrose gradients. The conditions used for labeling the protoplasts did not cause protoplast rupture or loss of viability. The distribution of the biotin label in these linear sucrose gradients was analyzed and compared to the distribution of vanadate-sensitive ATPase activity, a marker for the plasma membrane. Both the biotin label and the vanadate-sensitive ATPase activity were strongly localized in the gradient at peak density of 1.16 gram per cubic centimeter. When the protoplast surface was labeled, biotinylated polypeptides were detected after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and polypeptides of 153, 94, 51, 30, 20, 17, and 14 kilodaltons were shown to be plasma membrane in origin. When a crude membrane pellet was labeled...

Effect of Temperature on the Plasma Membrane and Tonoplast ATPases of Barley Roots 1: Comparison of Results Obtained with Acridine Orange and Quinacrine

DuPont, Frances M.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /04/1989 EN
Relevância na Pesquisa
574.88438%
The effect of temperature on the rate of proton transport and ATP hydrolysis by plasma membrane (PM) and tonoplast (TN) ATPases from barley (Hordeum vulgare L. cv CM 72) roots were compared. Rates of proton transport were estimated using the fluorescent amine dyes quinacrine and acridine orange. The ratio between rate of transport and ATP hydrolysis was found to depend on the dye, the temperature, and the type of membrane. The PM ATPase had an estimated Arrhenius energy of activation (Ea) of approximately 18 kilocalories per mole for ATP hydrolysis, and the Ea for proton transport was best estimated with acridine orange, which gave an Ea of 19 kilocalories per mole. The TN ATPase had an Ea for ATP hydrolysis of approximately 10 kilocalories per mole and the Ea for proton transport was best estimated with quinacrine, which gave an Ea of 10 kilocalories per mole. Acridine orange did not give an accurate estimate of Ea for the TN ATPase, nor did quinacrine for the PM ATPase. Reasons for the differences are discussed. Because it was suggested (AJ Pope, RA Leigh [1988] Plant Physiol 86: 1315-1322) that acridine orange interacts with anions to dissipate the pH gradient in TN vesicles, the complex effects of NO3− on the TN ATPase were also examined using acridine orange and quinacrine and membranes from oats and barley. Fluorescent amine dyes can be used to evaluate the effects of ions...

Functional Organization of Chlorophyll a and Carotenoids in the Alga, Nannochloropsis salina1

Brown, Jeanette S.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /02/1987 EN
Relevância na Pesquisa
574.78176%
Chlorophyll-protein complexes were isolated from a yellow-green alga, Nannochloropsis salina after mild detergent treatment and gel electrophoresis. Three different complexes were obtained which correspond to the three major kinds of chlorophyll-proteins isolated from spinach chloroplasts by the same procedure and previously identified as reaction center complexes for photosystems I and II and a light-harvesting complex. The analogy between the algal complexes and those from spinach was drawn from their absorption and fluorescence spectra and relative pigment content. The identities and amounts of the major carotenoids associated with each isolated complex were determined by HPLC. Although the reaction center complexes accounted for only 14% of the total chlorophyll, they were highly enriched in β-carotene, whereas the light-harvesting complex contained a high proportion of xanthophylls (mainly violaxanthin and vaucheriaxanthin-ester). Fluorescence excitation spectra of the algal membranes showed that one or both of the major xanthophylls may act as antenna pigment for photosynthesis.

Chlorophyll-Protein Complexes from the Red-Tide Dinoflagellate, Gonyaulax polyedra Stein 1: Isolation, Characterization, and the Effect of Growth Irradiance on Chlorophyll Distribution

Boczar, Barbara A.; Prezelin, Barbara B.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /04/1987 EN
Relevância na Pesquisa
574.91016%
A comparision of high (330 microeinsteins per meter squared per second) and low (80 microeinsteins per meter squared per second) light grown Gonyaulax polyedra indicated a change in the distribution of chlorophyll a, chlorophyll c2, and peridinin among detergent-soluble chlorophyll-protein complexes. Thylakoid fractions were prepared by sonication and centrifugation. Chlorophyll-protein complexes were solubilized from the membranes with sodium dodecyl sulfate and resolved by Deriphat electrophoresis. Low light cells yielded five distinct chlorophyll-protein complexes (I to V), while only four (I′ to IV′) were evident in preparations of high light cells. Both high molecular weight complexes I and I′ were dominated by chlorophyll a absorption and associated with minor amounts of chlorophyll c. Both complexes II and II′ were chlorophyll a-chlorophyll c2-protein complexes devoid of peridinin and unique to dinoflagellates. The chlorophyll a:c2 molar ratio of both complexes was 1:3, indicating significant chlorophyll c enrichment over thylakoid membrane chlorophyll a:c ratios of 1.8 to 2:1. Low light complex III differed from all other high or low light complexes in that it possessed peridinin and had a chlorophyll a:c2 ratio of 1:1. Low light complexes IV and V and high light complexes III′ and IV′ were spectrally similar...

Inhibition and Labeling of the Plant Plasma Membrane H+-ATPase with N-Ethylmaleimide 1

Katz, Donald B.; Sussman, Michael R.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /04/1987 EN
Relevância na Pesquisa
579.14613%
H+-ATPase activity in plasma membranes isolated from Avena sativa root cells is inhibited by N-ethylmaleimide, a covalent modifier of protein sulfhydryl groups. The rate of inhibition is reduced by ADP, MgADP, and MgATP, but even at 40 millimolar ADP the enzyme is only partially protected against inactivation. When plasma membranes are treated wth N-[2-3H]ethylmaleimide and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, prominent radioactive bands appear at Mr=100,000 and several other positions. However, only radioactivity in the Mr=100,000 protein is reduced by the presence of MgADP. These results provide independent evidence that the Mr=100,000 polypeptide which is observed in purified preparations of the enzyme is the catalytic subunit of the H+-ATPase. When tryptic peptides are produced from N-[2-3H]ethylmaleimide labeled Mr=100,000 protein and separated by reverse phase high performance liquid chromatography, two radioactive peaks are observed for which N-[2-3H]ethylmaleimide incorporation is reduced in the presence of MgADP.

b-Type Cytochromes in Higher Plant Plasma Membranes 1

Asard, Han; Venken, Mireille; Caubergs, Roland; Reijnders, Willem; Oltmann, Fred L.; De Greef, Jan A.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /07/1989 EN
Relevância na Pesquisa
579.14613%
The composition and characteristics of b-type cytochromes from higher plant plasma membranes, purified using aqueous two-phase partitioning, were investigated. At least three different cytochromes were identified by their wavelength maxima and redox midpoint potentials (E0′). Cytochrome b-560.7 (E0′ from + 110 to + 160 millivolts) was present in zucchini (Cucurbita pepo) hypocotyls and bean (Phaseolus vulgaris L.) hooks, although in different concentrations. The main component in cauliflower (Brassica oleracea L.) inflorescences (cytochrome b-558.8) is probably functionally similar to this cytochrome. The plasma membrane generally contains two to three cytochrome species. However, the occurrence and concentrations were species dependent. The high potential cytochrome can be reduced by ascorbate but not NADH, and may be involved in blue light perception.

Comparison of the Lipid Composition of Oat Root and Coleoptile Plasma Membranes 1: Lack of Short-Term Change in Response to Auxin

Sandstrom, Richard P.; Cleland, Robert E.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /07/1989 EN
Relevância na Pesquisa
579.00406%
The total lipid composition of plasma membranes (PM), isolated by the phase partitioning method from two different oat (Avena sativa L.) tissues, the root and coleoptile, was compared. In general, the PM lipid composition was not conserved between these two organs of the oat seedling. Oat roots contained 50 mole percent phospholipid, 25 mole percent glycolipid, and 25 mole percent free sterol, whereas comparable amounts in the coleoptile were 42, 39, and 19 mole percent, respectively. Individual lipid components within each lipid class also showed large variations between the two tissues. Maximum specific ATPase activity in the root PM was more than double the activity in the coleoptile. Treatment of coleoptile with auxin for 1 hour resulted in no detectable changes in PM lipids or extractable ATPase activity. Differences in the PM lipid composition between the two tissues that may define the limits of ATPase activity are discussed.

Purification of an H+-Translocating Inorganic Pyrophosphatase from Vacuole Membranes of Red Beet 1

Sarafian, Vahé; Poole, Ronald J.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /09/1989 EN
Relevância na Pesquisa
578.51688%
An H+-translocating inorganic pyrophosphatase (PPase) was isolated and purified from red beet (Beta vulgaris L.) tonoplast. One major polypeptide of molecular weight 67 kilodalton copurified with fluoride-inhibitable PPase activity when subjected to one-dimensional polyacrylamide gel electrophoresis. Overall, a 150-fold purification of the PPase was obtained, from the tonoplast fraction, through anion exchange chromatography of the detergent-solubilized membranes followed by ammonium sulfate precipitation and gel filtration chromatography. The purified polypeptide showed no cross-reactivity with antibodies raised against the 67 kilodalton subunit of the tonoplast ATPase.

Purification and Identification of a Plasma Membrane Associated Electron Transport Protein from Maize (Zea mays L.) Roots

Luster, Douglas G.; Buckhout, Thomas J.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /11/1989 EN
Relevância na Pesquisa
578.04492%
Plasma membranes isolated from three-day-old maize (Zea mays L.) roots by aqueous two-phase partitioning were used as starting material for the purification of a novel electron transport enzyme. The detergent-solubilized enzyme was purified by dyeligand affinity chromatography on Cibacron blue 3G-A-agarose. Elution was achieved with a gradient of 0 to 30 micromolar NADH. The purified protein fraction exhibited a single 27 kilodalton silver nitrate-stained band on sodium dodecyl sulfate polyacrylamide gel electrophoretograms. Staining intensity correlated with the enzyme activity profile when analyzed in affinity chromatography column fractions. The enzyme was capable of accepting electrons from NADPH or NADH to reduce either ferricyanide, juglone, duroquinone, or cytochrome c, but did not transfer electrons to ascorbate free-radical or nitrate. The high degree of purity of plasma membranes used as starting material as well as the demonstrated insensitivity to mitochondrial electron transport inhibitors confirmed the plasma membrane origin of this enzyme. The purified reductase was stimulated upon prolonged incubation with flavin mononucleotide suggesting that the enzyme may be a flavoprotein. Established effectors of plasma membrane electron transport systems had little effect on the purified enzyme...

Uptake and Accumulation of the Herbicide Bentazon by Cultured Plant Cells 1

Sterling, Tracy M.; Balke, Nelson E.; Silverman, Daniel S.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /04/1990 EN
Relevância na Pesquisa
579.00406%
Cellular absorption of the herbicide bentazon, a weak acid with pKa 3.45, was investigated using suspension-cultured cells of velvetleaf (Abutilon theophrasti Medic.). Bentazon accumulated rapidly to concentrations approximately four times that of the external medium. Bentazon accumulation against a concentration gradient was not due to its conversion to metabolites, partitioning into lipids, or binding onto cellular constituents. Bentazon uptake was related linearly to the external bentazon concentration, implying that movement of the herbicide into cells was not carrier-mediated. Bentazon was able to diffuse freely and extensively out of the cells, indicating that bentazon can readily diffuse across cell membranes. Potassium cyanide and carbonyl cyanide m-chlorophenyl hydrazone inhibited bentazon accumulation as did nitrogen gas when bubbled through the uptake medium. Absorption was pH-dependent with the greatest amount of bentazon accumulating at acidic external pH. Calculations indicated that conversion of uncharged bentazon to bentazon anion in the cytoplasm accounts for cellular accumulation of bentazon. These results provide evidence that bentazon is absorbed across membranes via simple diffusion and that bentazon accumulates in plant cells via an energy-dependent...

Age-Related Changes in Petal Membranes from Attached and Detached Rose Flowers

Itzhaki, Hannan; Borochov, Amihud; Mayak, Shimon
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /11/1990 EN
Relevância na Pesquisa
574.78176%
Changes in petal membrane properties during aging were studied in cut and in attached rose flowers (Rosa hybrida L., cv Mercedes). Both cut and attached flowers exhibited a growth phase characterized by an increase in fresh weight and an accumulation of membrane components. The growth phase, which was more pronounced in the attached than in the cut flowers, was followed by a senescence phase, characterized by a decrease in fresh weight and a decline in membrane components. In cut flowers, both the growth and the senescence phases were accompanied by a decrease in membrane fluidity and in the ratio of unsaturated to saturated fatty acids, but the ratio of sterol to phospholipid increased. In attached flowers, while both the membrane fluidity and the sterol-to-phospholipid ratio remained unchanged during the growth phase, the senescence phase was accompanied (as in cut flowers) by a decrease in membrane fluidity and an increase in the sterol-to-phospholipid ratio. Unlike in cut flowers, however, the age-related changes in the ratio of unsaturation of fatty acids were not correlated with those of fluidity. Changes in the saturation of phospholipid acyl chains are commonly thought to influence membrane fluidity. Our observations question this view and suggest instead that the ratio of sterol to phospholipid may play the major role in maintaining membrane lipid fluidity.

Na+/H+ and K+/H+ Antiport in Root Membrane Vesicles Isolated from the Halophyte Atriplex and the Glycophyte Cotton 1

Hassidim, Miriam; Braun, Yael; Lerner, Henri R.; Reinhold, Leonora
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /12/1990 EN
Relevância na Pesquisa
574.82113%
Proton fluxes have been followed into and out of membrane vesicles isolated from the roots of the halophyte Atriplex nummularia and the glycophyte Gossypium hirsutum, with the aid of the ΔpH probe [14C]methylamine. Evidence is presented for the operation of Na+/H+ and K+/H+ antiporters in the membranes of both plants. Cation supply after a pH gradient has been set up across the vesicle membrane (either as a result of providing ATP to the H+-ATPase or by imposing an artificial pH gradient) brings about dissipation of the ΔpH, but does not depolarize the membrane potential as observed in similar experiments, but in the absence of Cl−, using the ΔΨ probe SCN−. Cation/H+ exchange is thus indicated. This exchange is not due to nonspecific electric coupling, nor to competition for anionic adsorption sites on the membrane, nor to inhibition of the H+-ATPase; coupling of the opposed cation and H+ fluxes by a membrane component is the most likely explanation. Saturation kinetics have been observed for both Na+/H+ and K+/H+ antiport in Atriplex. Moreover, additive effects are obtained when Na+ is supplied together with saturating concentrations of K+, and vice versa, suggesting that separate antiporters for Na+ and for K+ may be operating. In the case of both Atriplex and Gossypium evidence was obtained suggesting the presence of antiporters in both plasmalemma and tonoplast.

Water Transport across Maize Roots 1: Simultaneous Measurement of Flows at the Cell and Root Level by Double Pressure Probe Technique

Zhu, Guo Li; Steudle, Ernst
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /01/1991 EN
Relevância na Pesquisa
574.78176%
A double pressure probe technique was used to measure simultaneously water flows and hydraulic parameters of individual cells and of excised roots of young seedlings of maize (Zea mays L.) in osmotic experiments. By following initial flows of water at the cell and root level and by estimating the profiles of driving forces (water potentials) across the root, the hydraulic conductivity of individual cell layers was evaluated. Since the hydraulic conductivity of the cell-to-cell path was determined separately, the hydraulic conductivity of the cell wall material could be evaluated as well (Lpcw = 0.3 to 6.10−9 per meter per second per megapascal). Although, for radial water flow across the cortex and rhizodermis, the apoplasmic path was predominant, the contribution of the hydraulic conductance of the cell-to-cell path to the overall conductance increased significantly from the first layer of the cortex toward the inner layers from 2% to 23%. This change was mainly due to an increase of the hydraulic conductivity of the cell membranes which was Lp = 1.9.10−7 per meter per second per megapascal in the first layer and Lp = 14 to 9.10−7 per meter per second per megapascal in the inner layers of the cortex. The hydraulic conductivity of entire roots depended on whether hydrostatic or osmotic forces were used to induce water flows. Hydrostatic Lpr was 1.2 to 2.3.10−7 per meter per second per megapascal and osmotic Lpr = 1.6 to 2.8.10−8 per meter per second per megapascal. The apparent reflection coefficients of root cells (σs) of nonpermeating solutes (KCI...

Identification and Characterization of Lipoxygenase Isoforms in Senescing Carnation Petals

Rouet-Mayer, Marie-Aude; Bureau, Jean-Marc; Laurière, Christiane
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /03/1992 EN
Relevância na Pesquisa
579.2536%
A membrane-associated lipoxygenase and a soluble lipoxygenase have been identified in carnation (Dianthus caryophyllus L. cv Rêve) petals. Treatments of microsomal membranes by nonionic or zwitterionic detergents indicated that lipoxygenase is tightly bound to the membranes. By phase separation in Triton X-114, microsomal lipoxygenase can be identified in part as an integral membrane protein. Soluble lipoxygenase had an optimum pH range of 4.9 to 5.8, whereas microsomal lipoxygenase exhibited maximum activity at pH 6.1. Both soluble and membrane-associated lipoxygenases produced carbonyl compounds and hydroperoxides simultaneously, in the presence of oxygen. The membranous enzyme was fully inhibited by 0.1 millimolar n-propyl gallate, nordihydroguaiaretic acid, or salicylhydroxamic acid, but the effect of the three inhibitors on the soluble enzyme was much lower. The soluble lipoxygenase is polymorphic and three isoforms greatly differing by their isoelectric points were identified. Lipoxygenase activity in flowers was maximal at the beginning of withering, both in the microsomal and the soluble fractions. Substantial variations in the ratio of the two forms of lipoxygenase were noted at different sampling dates. Our results allowed us to formulate the hypothesis of a strong association of one soluble form with defined membrane constituents.

Dissociation and Reassembly of the Vacuolar H+-ATPase Complex from Oat Roots 1

Ward, John M.; Reinders, Anke; Hsu, Hei-Ti; Sze, Heven
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /05/1992 EN
Relevância na Pesquisa
574.78176%
Conditions for the dissociation and reassembly of the multi-subunit vacuolar proton-translocating ATPase (H+-ATPase) from oat roots (Avena sativa var Lang) were investigated. The peripheral sector of the vacuolar H+-ATPase is dissociated from the membrane integral sector by chaotropic anions. Membranes treated with 0.5 molar KI lost 90% of membrane-bound ATP hydrolytic activity; however, in the presence of Mg2+ and ATP, only 0.1 molar KI was required for complete inactivation of ATPase and H+-pumping activities. A high-affinity binding site for MgATP (dissociation constant = 34 micromolar) was involved in this destabilization. The relative loss of ATPase activity induced by KI, KNO3, or KCl was accompanied by a corresponding increase in the peripheral subunits in the supernatant, including the nucleotide-binding polypeptides of 70 and 60 kilodaltons. The order of effectiveness of the various ions in reducing ATPase activity was: KSCN > KI > KNO3 > KBr > K-acetate > K2SO4 > KCl. The specificity of nucleotides (ATP > GTP > ITP) in dissociating the ATPase is consistent with the participation of a catalytic site in destabilizing the enzyme complex. Following KI-induced dissociation of the H+-ATPase, the removal of KI and MgATP by dialysis resulted in restoration of activity. During dialysis for 24 hours...

Proton Transport and Phosphorylation of Tonoplast Polypeptides from Zucchini Are Stimulated by the Phospholipid Platelet-Activating Factor 1

Martiny-Baron, Georg; Manolson, Morris F.; Poole, Ronald J.; Hecker, Doris; Scherer, Günther F. E.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /08/1992 EN
Relevância na Pesquisa
579.2536%
The ether phospholipid platelet-activating factor and certain similar phospholipids, including lysophosphatidylcholine, are known to stimulate both H+ transport and protein phosphorylation in plant microsomal membranes. In the present work, several polypeptides in highly purified tonoplast membranes from zucchini (Cucurbita pepo L.) showed platelet-activating factor-dependent phosphorylation. Comparison of protein phosphorylation in different membrane fractions separated by sucrose step density gradient centrifugation indicated that some of the phosphoproteins were contaminants or were common to several membrane fractions, but platelet-activating factor-dependent phosphorylation of peptides at 30, 53, and perhaps 100 kilodaltons was tonoplast specific. The phosphoprotein of 53 kilodaltons was shown by three different approaches (one- and two-dimensional polyacrylamide gel electrophoresis, western blots, and immunoprecipitation) to cross-react with antibody raised against the B subunit of the tonoplast ATPase from red beet (Beta vulgaris L.).

Evidence for and Subcellular Localization of a Ca-Stimulated Phospholipase D from Maize Roots

Brauer, David; Nungesser, Edwin; Maxwell, Robert J.; Schubert, Carol; Tu, Shu-l
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /03/1990 EN
Relevância na Pesquisa
579.2536%
Autolytic lipid changes in corn (Zea mays L.) root crude homogenates and isolated membranes were examined by the use of high performance thin-layer chromatography. In the absence of added CaCl2, losses in phosphatidylcholine and other phospholipids corresponds to increase in fatty acids without the accumulation of either phosphatidic acid or lyso-phosphatidylcholine. However, in the presence of 1 millimolar CaCl2, phosphatidylcholine concentrations declined more rapidly with an immediate increase in phoshatidic acid, and slower rate of fatty acid accumulation. Autolytic phospholipid degradation yielded primarily free fatty acids in the absence of Ca and phosphatidic acid in the presence of 1 millimolar CaCl2, suggesting the presence of an acyl hydrolase and phospholipase D activities. Differential centrifugation studies indicate that 50 to 80% of the crude homogenate's phospholipase D activity is membrane-bound. Density centrifugation experiments suggest that the membrane-bound phospholipase D activity is localized primarily on mitochondrial membranes.