Martiny-Baron G, Scherer G F
Botanisches Institut der Universität Bonn, Federal Republic Germany.
J Biol Chem. 1989 Oct 25;264(30):18052-9.
In membrane fractions from zucchini (Cucurbita pepo L.) hypocotyls, catalytic properties of a platelet-activating factor (PAF)-activated protein kinase were investigated. In the presence of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid, phosphorylation of a 55-kDa membrane polypeptide and, to a lesser extent, several others, including a 120-kDa polypeptide, was stimulated by PAF. The phosphorylation of the 55-kDa polypeptide was used for quantification of the PAF-stimulated protein kinase. Stimulation of protein phosphorylation by PAF increased in a concentration range from 10-200 micrograms/ml (= 19-380 microM) PAF up to 10-fold above the control. Addition of Ca2+ ions in the micromolar range in the presence and in the absence of PAF increased the phosphorylation of the 55- and the 120-kDa polypeptide. Other phospholipids and lipids tested including phorbol ester, diglyceride, mono- and triglyceride, and oleic acid were ineffective. The same lipid specificity was previously observed for the activation of ATP-dependent H+ transport in microsomes (Scherer, G.F.E., Martiny-Baron, G., and Stoffel, B. (1988) Planta 175, 241-253). Lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE) were able to stimulate the phosphorylation of the same polypeptides as PAF and H+ transport but both to a lesser extent (PAF greater than LPC greater than LPE). In the presence of EGTA, PAF-stimulated phosphorylation of a 55- and a 57-kDa polypeptide was predominantly associated with vacuolar membranes and those of 42, 61, 63, and 120 kDa were predominantly associated with plasma membranes. Stimulation of ATP-dependent H+ transport by PAF was found in tonoplast vesicles whereas plasma membrane vesicles had only little transport activity and, therefore, an effect of PAF on plasma membrane H+ transport could not be measured. Stimulation of ATP hydrolysis by PAF was observed both in tonoplast- and plasma membrane-containing fractions.
在西葫芦（南瓜属西葫芦种）下胚轴的膜组分中，研究了血小板活化因子（PAF）激活的蛋白激酶的催化特性。在[乙二胺双（氧乙烯腈）]四乙酸存在的情况下，PAF刺激了一条55 kDa膜多肽的磷酸化，在较小程度上也刺激了包括一条120 kDa多肽在内的其他几条多肽的磷酸化。55 kDa多肽的磷酸化用于定量PAF刺激的蛋白激酶。PAF对蛋白磷酸化的刺激在10 - 200微克/毫升（= 19 - 380微摩尔）的PAF浓度范围内增加，比对照高10倍。在有和没有PAF的情况下，添加微摩尔浓度的Ca²⁺离子都会增加55 kDa和120 kDa多肽的磷酸化。测试的其他磷脂和脂质，包括佛波酯、甘油二酯、甘油单酯和甘油三酯以及油酸，均无效。之前在微粒体中ATP依赖的H⁺转运激活中也观察到了相同的脂质特异性（Scherer, G.F.E., Martiny - Baron, G., and Stoffel, B. (1988) Planta 175, 241 - 253）。溶血磷脂酰胆碱（LPC）和溶血磷脂酰乙醇胺（LPE）能够刺激与PAF和H⁺转运相同的多肽的磷酸化，但程度都较小（PAF＞LPC＞LPE）。在EGTA存在的情况下，PAF刺激的55 kDa和57 kDa多肽的磷酸化主要与液泡膜相关，而42、61、63和120 kDa的磷酸化主要与质膜相关。在液泡膜小泡中发现PAF刺激了ATP依赖的H⁺转运，而质膜小泡的转运活性很小，因此无法测量PAF对质膜H⁺转运的影响。在含有液泡膜和质膜的组分中均观察到PAF刺激的ATP水解。
