Liposomal membranes. 13. Transport of an amino acid across liposomal bilayers as mediated by a photoresponsive carrier
Liposomal membranes. 13. Transport of an amino acid across liposomal bilayers as mediated by a photoresponsive carrier
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DOI：
10.1021/ja00384a049
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发表时间：
1982-10
影响因子：
15
通讯作者：
J. Sunamoto;K. Iwamoto;Yukio Mohri;T. Kominato
中科院分区：
文献类型：
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作者：
J. Sunamoto;K. Iwamoto;Yukio Mohri;T. Kominato
0卵PC的总浓度为1.0mM，外部的pH保持恒定在8.5。箭头指示运输方向。B在UV照射20分钟和紫外光照射5分钟后测定Phe-OMe的转运量。开环和闭环物质可溶于非极性溶剂如己烷和辛醇中，这从以下事实中显而易见，即使当含有1的己烷溶液与缓冲水溶液一起振荡时（pH6.0）在25 ℃下UV照射10分钟后，紫色开环物质仍保留在己烷层中。预期所得两性离子染料2（与中性形式1相反）与两性离子氨基酸形成离子络合物。当然，当苯丙氨酸（Phe）或其甲酯（Phe-OMe）置于水层中时，在紫外光照射下，氨基酸从水相转移到有机相。10这通过使用荧光胺荧光测定水相氨基酸浓度的降低来检查。“当然，在黑暗中或不存在1的情况下，没有观察到氨基酸的分布。Phe比Phe-OMe更有效地被2转运，这揭示了2和氨基酸之间的离子缔合的重要性，而不是疏水作用。这些预先调查的光控转移的氨基酸从水相的有机相含有photospiran 1鼓励我们尝试类似的运输的氨基酸通过脂质体膜介导的2。当用脂质体膜代替液膜时，有必要确定在光照射下通过脂质的光化学裂解或通过光螺旋素包埋的双层的物理化学无序诱导氨基酸泄漏的可能性。在照射期间和照射后，均未观察到脂质体悬浮液的pH值变化伴随着α-磷脂酰胆碱头基的破坏，也未观察到来自卵磷脂的任何降解产物。这通过脂质体悬浮液的直接pH测量和通过从Sephadex G-50柱洗脱的经辐照的脂质体悬浮液的重复凝胶过滤来证实。为了检测由包埋在脂质体双层中的1的光致变色引起的脂质体双层的可能的物理化学无序，我们在脂质体内部封装了水溶性荧光探针，吡喃（8-羟基-1，3，6-芘三磺酸三钠），而不是氨基酸。[12]由于预计吡喃不能与2形成任何稳定的络合物，因此在光照射下吡喃的泄漏只能由双层的物理化学无序引起。然而，在UV和可见光照射下未观察到吡喃碱泄漏。（10）在25 ℃下紫外线照射10分钟后，将1.5-mL含有1 × 10 ~（TM）4 M氨基酸的水性缓冲溶液（pH 6.0）的混合物与1.5 mL含有1 × 10 ~（TM）3 L的己烷在涡旋混合器上剧烈振荡。根据文献（参考文献11）中描述的方法，立即用荧光胺荧光测定水相中的氨基酸浓度。通过该程序，30.0%的Phe和20.3%的Phe-OMe分别被2从水相转移到有机相。
0 Total concentration of egg PC was 1.0 mM. The pH of the ex-terior was kept constant at 8.5. An arrow indicates the direction of transport. b Amounts of Phe-OMe transported were determined after UV irradiation for 20 min and visiblelight irradiation for 5 min. ring-opened and-closed species are soluble in apolar solvents such as hexane and octanol, and this is evident from the fact that even when the hexane solution containing 1 was shaken with an aqueous buffered solution (pH 6.0) after UV irradiation for 10 min at 25 C, the purple ring-opened species still remained in the hexane layer. The resulting zwitterionic dye2 (in contrast to the neutral form 1) is expected toform an ionic complex with a zwitterionic-amino acid. Certainly, when phenylalanine (Phe) or its methyl ester (Phe-OMe) was placed in the water layer, the amino acid was transfered from the water phase to theorganic phase upon UV irradiation. 10 This was examined by determining the decrease in the amino acid concentration of the aqueous phase fluorome-trically by using fluorescamine." Of course, no distribution of amino acid was observed in the dark or in the absence of 1. Phe was more effectively transported by 2 than was Phe-OMe, and this revealed the greater importance of the ionic association between 2 and the amino acid as opposed tohydrophobic effects. These preinvestigations on a photocontrolled transfer of an amino acid from the water phase to the organic phase containing the photospiran 1 encouraged us to try the similar transport of an amino acid across liposomal membranes as mediated by 2. When a liposomal membrane is employed instead of a liquid membrane, it is necessaryto ascertain the improbability of induced leakage of amino acid either by photochemical lysis of lipids or by physicochemical disordering of the photospiran-embedded bilayers upon photoirradiation. Neither a pH change of the liposome suspension accompanied by destruction of the phos-phatidylcholine head group nor any degradates derived from lecithins was observed during and after the irradiation. This was confirmed by direct pH measurement of the liposome suspension and by repeated gel filtration of the irradiated liposome suspensions eluted from a Sephadex G-50 column. In order to detect possible physicochemical disordering of liposomal bilayers as caused by the photochromism of 1 embedded in the bilayers, we encapsulated a water-soluble fluorescent probe, pyranine (trisodium 8-hydroxy-1, 3, 6-pyrenetrisulfonate), instead of amino acid in the interior of the liposomes. 12 Since pyranine is not expected to form any stable complex with 2, the leakage of pyranine under photoirradiation must result only from the physicochemical disordering of the bilayers. However, no leakage of pyranine under UV and visible light irradiation was observed. 13 (10) A 1.5-mL mixture of aqueous buffered solution (pH 6.0) containing 1 X 10™ 4 M aminoacid was vigorously shaken with 1.5 mL of hexane con-taining 1 X 10™ 3 1 on a Vortex mixer after UV irradiation for 10 min at 25 C. The amino acid concentration in thewater phase was immediately determined fluorometrically with fluorescamine according to the method described in the literature (ref 11). By this procedure, 30.0% Phe and 20.3% Phe-OMe were transported by 2, respectively, from the aqueous phase to the organic phase.