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首页> 外文期刊>Protoplasma: An International Journal of Cell Biology >Transport of ascorbate into protoplasts of Nicotiana tabacum Bright Yellow-2 cell line
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Transport of ascorbate into protoplasts of Nicotiana tabacum Bright Yellow-2 cell line

机译：抗坏血酸转运至烟草亮黄2细胞系的原生质体中

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The uptake of ascorbate into protoplasts isolated from a Nicotiana tabacum Bright Yellow-2 (BY-2) cell suspension culture was investigated. Addition of C-14-labelled ascorbate to freshly isolated protoplasts resulted in a time- and substrate-dependent association of radioactive molecules with the protoplasts. The kinetic characterisation of this presumptive uptake revealed kinetics of Michaelis-Menten type with an apparent maximal uptake activity of 24 pmol/min . 10(6) protoplasts and an apparent affinity constant of 139 mu M. The amount of ascorbate molecules transported into N. tabacum protoplasts decreased when nonlabelled dehydroascorbate or iso-ascorbate were added but was not affected by addition of 5,6-o-cyclohexylidene ascorbate or ascorbate-2-sulfate. These data indicate a carrier-mediated uptake of ascorbate into the protoplasts that shows a high structural specificity. To investigate which redox status of ascorbate is preferentially taken up by the N. tabacum protoplasts, transport was rested in the presence of various compounds that can affect the redox status of ascorbate. Testing uptake in the presence of a reductant, dithiothreitol, resulted in a significant and concentration-dependent inhibition of the amount of ascorbate molecules transported into the protoplasts. On the other hand, ascorbate uptake was significantly stimulated in the presence of the enzyme ascorbate oxidase. Ferricyanide did not affect ascorbate transport. Inhibition studies revealed that ascorbate uptake in the protoplasts is sensitive to addition of sulfhydryl reagents N-ethyl maleimide and p-chloromercuribenzenesulfonic acid and to a disruption of the proton gradient by the protonophore carbonylcyanide-3-chlorophenylhydrazone. The uptake of ascorbate was also inhibited by addition of cytochalasin B but not sensitive to addition of phloretin or sulfinpyrazone. Taken together these data indicate the presence of an ascorbate transport system in the plasma membrane of N, tabacum protoplasts and suggest dehydroascorbate as the preferentially transported redox species. The putative presence of different carriers for reduced and oxidised ascorbate in the plasma membrane is discussed. [References: 32]
机译：研究了从烟草（Nicotiana tabacum）亮黄色2（BY-2）细胞悬浮培养物中分离出的原生质体中抗坏血酸的吸收。将C-14标记的抗坏血酸盐添加到新鲜分离的原生质体中会导致放射性分子与原生质体发生时间和底物依赖性的结合。该推测摄取的动力学特征揭示了Michaelis-Menten型动力学，其表观最大摄取活性为24 pmol/min。 10（6）原生质体，表观亲和常数为139μM。当添加未标记的脱氢抗坏血酸或异抗坏血酸时，转运到烟草原生质体中的抗坏血酸分子的数量减少，但不受5，6-o-环己二烯的影响抗坏血酸或2-硫酸抗坏血酸。这些数据表明载体介导的抗坏血酸对原生质体的吸收，具有很高的结构特异性。为了研究烟草的原生质体优先吸收哪种抗坏血酸的氧化还原状态，在各种可能影响抗坏血酸的氧化还原状态的化合物存在下进行转运。在还原剂二硫苏糖醇的存在下测试摄取量，会导致对转运至原生质体中的抗坏血酸分子数量的抑制作用显着且浓度依赖性。另一方面，在抗坏血酸氧化酶的存在下显着刺激了抗坏血酸的摄取。铁氰化物不影响抗坏血酸的转运。抑制研究表明，原生质体中抗坏血酸的吸收对巯基试剂N-乙基马来酰亚胺和对氯mercuri苯磺酸的添加敏感，并且对质子梯度的质子梯度羰基氰-3-氯苯基phenyl破坏质子梯度很敏感。抗坏血酸的摄取也受到细胞松弛素B的抑制，但对生螺菌素或亚砜并吡嗪不敏感。这些数据加在一起表明N，烟草原生质体的质膜中存在抗坏血酸转运系统，并建议将脱氢抗坏血酸作为优先转运的氧化还原物质。讨论了质膜中还原和氧化的抗坏血酸盐的不同载体的假定存在。 [参考：32]

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