Kasai M, Muto S
Institute of Applied Microbiology, University of Tokyo, Japan.
J Membr Biol. 1990 Mar;114(2):133-42. doi: 10.1007/BF01869094.
Plasma membrane vesicles, which are mostly right side-out, were isolated from corn leaves by aqueous two-phase partitioning method. Characteristics of Ca2+ transport were investigated after preparing inside-out vesicles by Triton X-100 treatment. 45Ca2+ transport was assayed by membrane filtration technique. Results showed that Ca2+ transport into the plasma membrane vesicles was Mg-ATP dependent. The active Ca2+ transport system had a high affinity for Ca2+(Km(Ca2+) = 0.4 microM) and ATP(Km(ATP) = 3.9 microM), and showed pH optimum at 7.5. ATP-dependent Ca2+ uptake in the plasma membrane vesicles was stimulated in the presence of Cl- or NO3-. Quenching of quinacrine fluorescence showed that these anions also induced H+ transport into the vesicles. The Ca2+ uptake stimulated by Cl- was dependent on the activity of H+ transport into the vesicles. However, carbonylcyanide m-chlorophenylhydrazone (CCCP) and VO4(3-) which is known to inhibit the H+ pump associated with the plasma membrane, canceled almost all of the Cl(-)-stimulated Ca2+ uptake. Furthermore, artificially imposed pH gradient (acid inside) caused Ca2+ uptake into the vesicles. These results suggest that the Cl(-)-stimulated Ca2+ uptake is caused by the efflux of H+ from the vesicles by the operation of Ca2+/H+ antiport system in the plasma membrane. In Cl(-)-free medium, H+ transport into the vesicles scarcely occurred and the addition of CCCP caused only a slight inhibition of the active Ca2+ uptake into the vesicles. These results suggest that two Ca2+ transport systems are operating in the plasma membrane from corn leaves, i.e., one is an ATP-dependent active Ca2+ transport system (Ca2+ pump) and the other is a Ca2+/H+ antiport system. Little difference in characteristics of Ca2+ transport was observed between the plasma membranes isolated from etiolated and green corn leaves.
通过水相两相分配法从玉米叶片中分离出大多为外翻的质膜囊泡。经Triton X - 100处理制备内翻囊泡后，研究了Ca2+转运特性。采用膜过滤技术测定45Ca2+转运。结果表明，Ca2+转运进入质膜囊泡依赖于Mg - ATP。活性Ca2+转运系统对Ca2+(Km(Ca2+) = 0.4 microM)和ATP(Km(ATP) = 3.9 microM)具有高亲和力，且在pH 7.5时表现出最佳活性。在Cl-或NO3-存在下，质膜囊泡中依赖ATP的Ca2+摄取受到刺激。喹吖因荧光猝灭表明这些阴离子也诱导H+转运进入囊泡。Cl-刺激的Ca2+摄取依赖于H+转运进入囊泡的活性。然而，羰基氰化物间氯苯腙(CCCP)和已知抑制与质膜相关的H+泵的VO4(3-)几乎消除了所有Cl(-)刺激的Ca2+摄取。此外，人为施加的pH梯度(内部为酸性)导致Ca2+摄取进入囊泡。这些结果表明，Cl(-)刺激的Ca2+摄取是由质膜中Ca2+/H+反向转运系统的运作使H+从囊泡外流引起的。在无Cl(-)培养基中，H+几乎不转运进入囊泡，添加CCCP仅对囊泡中活性Ca2+摄取产生轻微抑制。这些结果表明，玉米叶片的质膜中存在两种Ca2+转运系统，即一种是依赖ATP的活性Ca2+转运系统(Ca2+泵)，另一种是Ca2+/H+反向转运系统。从黄化和绿色玉米叶片分离的质膜之间，在Ca2+转运特性上未观察到显著差异。
