Effects of electric fields on the membrane enzymes of plant cells

点击翻译按钮获取中文摘要

The aims of the present project is to study the effects of external electric fields on the in vivo catalytic functions of the plasma-membrane enzyme, H-ATPase, of plant cells. We employed the patch clamp technique to monitor the proton-pumping activity of H-ATPase in the plasma membranes of plant cells. In order to be able to detect the proton-pumping activity as H current by the patch clamp technique, we must find the conditions under which the currents mediated by the ion channels are negligibly small compared with the H current mediated by H-ATPase in the plasma membrane. On the other hand, the membrane potentials of intact cells provide a useful indicator of in vivo activity of H-ATPase in their plasma membranes. Thus the present project needs to develop a convenient method for determinig the membrane potential of protoplasts. 1. The properties of ion channels in the plasma membrane of tobacco cultured protoplasts were studied in detail by using the patch clamp technique. It was sh … More own that two types of ion channels, K and C1 channels, are present in the plasma membrane, that these channels can open only in the depolarization range of the transmembrane potential, and that the order of increasing permeability of the K channel to alkali metal cations is Cs<Na-Li<K. When the transmembrane potential was clamped in the hyperpolarization range, addition of fusicoccin, which is known to activate the plasma membrane H-ATPase in plant cells, produced a transient outward current in whole-cell configuration. The characterization of this transient current is now in progress. 2. The theoretical expressions were derived for the relationship between the fluorescence response of the cyanine dye, 3,3'-dipropylthiadicarbocyanine (dis-C3-(5)), added to a cell suspension and the membrane potential of the cell on the basis of the mechanism suggested by Sims et al. The condions which should be satisfied in evaluating the membrane potential by this method are presented. The theoretical predictions agreed well with the experimental results obtained with protplasts isolated from Lithospermum erythrorhizon cultured cells. Less