Ca2+ pump and Ca2+/H+ antiporter in plasma membrane vesicles isolated by aqueous two-phase partitioning from corn leaves.
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.
Kasai
M
M
Institute of Applied Microbiology, University of Tokyo, Japan.
Muto
S
S
D000255:Adenosine Triphosphate / Q000502:physiology; D017920:Antiporters; D001693:Biological Transport, Active / Q000187:drug effects; D015220:Calcium Channels / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D027682:Cation Transport Proteins; D002462:Cell Membrane / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007811:Lanthanum / Q000494:pharmacology; D008274:Magnesium / Q000502:physiology; D011522:Protons; D012492:Salts / Q000494:pharmacology; D014638:Vanadates / Q000494:pharmacology; D014700:Verapamil / Q000494:pharmacology; D003313:Zea mays / Q000378:metabolism*
D017920:Antiporters; D015220:Calcium Channels; D002135:Calcium-Binding Proteins; D027682:Cation Transport Proteins; D011522:Protons; D012492:Salts; C027614:calcium-hydrogen antiporters; D014638:Vanadates; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D007811:Lanthanum; D000255:Adenosine Triphosphate; D014700:Verapamil; D008274:Magnesium