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- Askerlund, Per, et al.
(author)
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NADH-Ferricyanide Reductase of Leaf Plasma Membranes : Partial Purification and Immunological Relation to Potato Tuber Microsomal NADH-Ferricyanide Reductase and Spinach Leaf NADH-Nitrate Reductase
- 1991
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In: Plant Physiology. - 0032-0889 .- 1532-2548. ; 95:1, s. 6-13
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Journal article (peer-reviewed)abstract
- Plasma membranes obtained by two-phase partitioning of microsomal fractions from spinach (Spinacea oleracea L. cv Medania) and sugar beet leaves (Beta vulgaris L.) contained relatively high NADH-ferricyanide reductase and NADH-nitrate reductase (NR; EC 1.6.6.1) activities. Both of these activities were latent. To investigate whether these activities were due to the same enzyme, plasma membrane polypeptides were separated with SDS-PAGE and analyzed with immunoblotting methods. Antibodies raised against microsomal NADH-ferricyanide reductase (tentatively identified as NADH-cytochrome b5 reductase, EC 1.6.2.2), purified from potato (Solanum tuberosum L. cv Bintje) tuber microsomes, displayed one single band at 43 kilodaltons when reacted with spinach plasma membranes, whereas lgG produced against NR from spinach leaves gave a major band at 110 kilodaltons together with a few fainter bands of lower molecular mass. Immunoblotting analysis using inside-out and right-side-out plasma membrane vesicles strongly indicated that NR was not an integral protein but probably trapped inside the plasma membrane vesicles during homogenization. Proteins from spinach plasma membranes were solubilized with the zwitterionic detergent 3-[(3-cholamidopropyl) dimethylammonio] 1-propane-sulfonate and separated on a Mono Q anion exchange column at pH 5.6 with fast protein liquid chromatography. One major peak of NADH-ferricyanide reductase activity was found after separation. The peak fraction was enriched about 70-fold in this activity compared to the plasma membrane. When the peak fractions were analyzed with SDS-PAGE the NADH-ferricyanide reductase activity strongly correlated with a 43 kilodalton polypeptide which reacted with the antibodies against potato microsomal NADH-ferricyanide reductase. Thus, our data indicate that most, if not all, of the truly membrane-bound NADH-ferricyanide reductase activity of leaf plasma membranes is due to an enzyme very similar to potato tuber microsomal NADH-ferricyanide reductase (NADH-cytochrome b5 reductase).
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| 2. |
- Askerlund, Per, et al.
(author)
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Transmembrane Electron Transport in Plasma Membrane Vesicles Loaded with an NADH-Generating System or Ascorbate
- 1991
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In: Plant Physiology. - 0032-0889 .- 1532-2548. ; 96:4, s. 1178-1184
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Journal article (peer-reviewed)abstract
- Sugar beet (Beta vulgaris L.) leaf plasma membrane vesicles were loaded with an NADH-generating system (or with ascorbate) and were tested spectrophotometrically for their ability to reduce external, membrane-impermeable electron acceptors. Either alcohol dehydrogenase plus NAD+ or 100 millimolar ascorbate was included in the homogenization medium, and right-side-out (apoplastic side-out) plasma membrane vesicles were subsequently prepared using two-phase partitioning. Addition of ethanol to plasma membrane vesicles loaded with the NADH-generating system led to a production of NADH inside the vesicles which could be recorded at 340 nanometers. This system was able to reduce 2,6-dichlorophenolindophenol-3′-sulfonate (DCIP-sulfonate), a strongly hydrophilic electron acceptor. The reduction of DCIP-sulfonate was stimulated severalfold by the K+ ionophore valinomycin, included to abolish membrane potential (outside negative) generated by electrogenic transmembrane electron flow. Fe3+-chelates, such as ferricyanide and ferric citrate, as well as cytochrome c, were not reduced by vesicles loaded with the NADH-generating system. In contrast, right-side-out plasma membrane vesicles loaded with ascorbate supported the reduction of both ferric citrate and DCIP-sulfonate, suggesting that ascorbate also may serve as electron donor for transplasma membrane electron transport. Differences in substrate specificity and inhibitor sensitivity indicate that the electrons from ascorbate and NADH were channelled to external acceptors via different electron transport chains. Transplasma membrane electron transport constituted only about 10% of total plasma membrane electron transport activity, but should still be sufficient to be of physiological significance in, e.g. reduction of Fe3+ to Fe2+ for uptake.
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| 10. |
- Ekbom, L. B., et al.
(author)
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Liquid phase sintering of tungsten composites under microgravity : Influence of liquid/particle surface energy
- 1991
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In: Advances in Space Research. - 0273-1177 .- 1879-1948. ; 11:7, s. 331-335
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Journal article (peer-reviewed)abstract
- Tungsten-nickel-iron heavy metals are fabricated from powders by liquid phase sintering. A study of the sintering mechanism of heavy metals with a low amount of tungsten particles can, depending on sedimentation, only be done under microgravity. At two earlier Texus flights, No 10 & 19, the growth rate and agglomeration of tungsten particles under microgravity were compared to that under gravity and sedimentation. During the first minute sintering period the particles in the agglomerates were separated and the particle distribution became even. The particle growth rate under microgravity, initially high, is slowing down. At further studies under gravity the influence of surface energy between the liquid phase and the tungsten particles have been investigated. Additions of sulphur and cobolt were used to decrease the surface energy and the use of nickel without iron to increase the energy. At a high surface energy the growth rate increases due to increased driving force. Preliminary results from the Texus 26 flight (April 90) are included. © 1991.
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