Schmidt A L, Briskin D P
Department of Agronomy, University of Illinois, Urbana 61801.
Arch Biochem Biophys. 1993 Feb 15;301(1):165-73. doi: 10.1006/abbi.1993.1129.
The H+/substrate stoichiometries of the tonoplast H(+)-ATPase and H(+)-PPase were determined by a kinetic approach. Using red beet (Beta vulgaris L.) tonoplast vesicles, rates of substrate-dependent H+ transport were estimated by (I) a mathematical model describing the time course of delta pH formation, (II) the rate of H+ leakage following H+ pump inhibition at a steady state delta pH, and (III) the initial rate of alkalinization of the external medium. When compared with rates of substrate hydrolysis measured under identical conditions, all three methods yielded an H+/ATP stoichiometry of 2 while the H+/PPi stoichiometry was determined to be 1 using methods I and II. Experimental limitations did not permit an analysis of the H+/PPi stoichiometry by method III. From these results and the estimated level of substrate and product typically found in the cytoplasm of plant cells, it is suggested that the H(+)-ATPase and H(+)-PPase as primary H(+)-pumps are poised toward net substrate hydrolysis under in vivo conditions thereby operating in parallel to generate a proton electrochemical gradient across the tonoplast.
通过动力学方法测定了液泡膜H⁺-ATP酶和H⁺-PP酶的H⁺/底物化学计量比。使用红甜菜（Beta vulgaris L.）液泡膜囊泡，通过以下方法估算底物依赖性H⁺转运速率：（I）描述ΔpH形成时间进程的数学模型；（II）在稳定状态的ΔpH下H⁺泵抑制后H⁺泄漏速率；（III）外部介质碱化的初始速率。与在相同条件下测得的底物水解速率相比，所有三种方法得出的H⁺/ATP化学计量比均为2，而使用方法I和II确定的H⁺/PPi化学计量比为1。实验限制使得无法通过方法III分析H⁺/PPi化学计量比。根据这些结果以及植物细胞细胞质中通常发现的底物和产物估计水平，表明H⁺-ATP酶和H⁺-PP酶作为主要的H⁺泵，在体内条件下倾向于净底物水解，从而并行运作以在液泡膜上产生质子电化学梯度。
