Ferrol N., Belver A., Roldan M., Rodriguez-Rosales M. P., Donaire J. P.
Departamento de Bioquimica Vegetal, Estacion Experimental del Zaidin, Consejo Superior de Investigaciones Cientificas, 18008 Granada, Spain.
Plant Physiol. 1993 Nov;103(3):763-769. doi: 10.1104/pp.103.3.763.
Boron deficiency and toxicity inhibit ATP-dependent H+ pumping and vanadate-sensitive ATPase activity in sunflower roots and cell suspensions. The effects of boron on H+ pumping and on passive H+ conductance, as well as on fluorescence anisotropy in KI-washed microsomes isolated from sunflower (Helianthus annuus L. cv Enano) cell suspensions, have been investigated. Boron deficiency reduced the total and vanadate-sensitive ATPase activities as well as the vanadate-sensitive ATP-dependent H+ pumping without affecting the amount of antigenic ATPase protein as measured by immunoblotting with an Arabidopsis thaliana plasma membrane anti-H+-ATPase polyclonal antibody. Kinetic studies revealed that boron deficiency reduced Vmax of vanadate-sensitive ATPase activity with little change in the apparent Km for Mg2+-ATP. Proton leakage was greater in microsomal vesicles isolated from cells grown without boron and incubated in reaction medium without added boron, and this effect was reversed by addition of boron to the reaction medium. Fluorescence anisotropy indicated that diphenyl hexatriene and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene probes were immobilized to a greater extent in microsomes from cells grown without boron than in those from cells grown with 100 [mu]M H3BO3. The apparent decrease of membrane fluidity in microsomes from cells grown without boron was reversed by the addition of boron to the reaction medium. Taken together these data suggest that inhibition of H+ gradient formation in microsomes from sunflower cells grown in the absence of boron could be due to the combined effects of reduced H+-ATPase activity and increased passive conductance across the membrane, possibly resulting from increased membrane rigidity.
硼缺乏和毒性会抑制向日葵根系和细胞悬浮液中依赖ATP的H⁺泵浦以及钒酸盐敏感的ATP酶活性。研究了硼对从向日葵（Helianthus annuus L. cv Enano）细胞悬浮液中分离出的KI洗涤微粒体中H⁺泵浦、被动H⁺电导率以及荧光各向异性的影响。硼缺乏降低了总ATP酶活性和钒酸盐敏感的ATP酶活性，以及钒酸盐敏感的依赖ATP的H⁺泵浦，而用拟南芥质膜抗H⁺ - ATP酶多克隆抗体进行免疫印迹测量时，不影响抗原性ATP酶蛋白的量。动力学研究表明，硼缺乏降低了钒酸盐敏感的ATP酶活性的Vmax，而对Mg²⁺ - ATP的表观Km变化不大。从无硼培养的细胞中分离出并在无添加硼的反应介质中孵育的微粒体囊泡中质子泄漏更大，而向反应介质中添加硼可逆转这种效应。荧光各向异性表明，二苯基己三烯和1 -（4 - 三甲基铵苯基）- 6 - 苯基 - 1,3,5 - 己三烯探针在无硼培养的细胞的微粒体中比在含有100 μM H₃BO₃培养的细胞的微粒体中固定程度更高。向反应介质中添加硼可逆转无硼培养的细胞的微粒体中膜流动性的明显降低。综合这些数据表明，在无硼条件下生长的向日葵细胞的微粒体中H⁺梯度形成的抑制可能是由于H⁺ - ATP酶活性降低和跨膜被动电导率增加的综合作用，这可能是由于膜刚性增加所致。
