Veluthambi K, Poovaiah B W
Department of Horticulture and Landscape Architecture, Washington State University, Pullman 99164-6414, USA.
Plant Physiol. 1986 Jul;81(3):836-41. doi: 10.1104/pp.81.3.836.
In vitro and in vivo protein phosphorylations in oat (Avena sativa L.) coleoptile segments were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and by two-dimensional gel electrophoresis. In vitro phosphorylation of several polypeptides was distinctly promoted at 1 to 15 micromolar free Ca2+ concentrations. Ca2(+)-stimulated phosphorylation was markedly reduced by trifluoperazine, chlorpromazine, and naphthalene sulfonamide (W7). Two polypeptides were phosphorylated both under in vitro and in vivo conditions, but the patterns of phosphorylation of several other polypeptides were different under the two conditions indicating that the in vivo phosphorylation pattern of proteins is not truly reflected by in vitro phosphorylation studies. Trifluoperazine, W7, or ethylene glycol-bis-(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) + calcium ionophore A23187 treatments resulted in reduced levels of in vivo protein phosphorylation of both control and auxin-treated coleoptile segments. Analysis by two-dimensional electrophoresis following in vivo phosphorylation revealed auxin-dependent changes of certain polypeptides. A general inhibition of phosphorylation by calmodulin antagonists suggested that both control and auxin-treated coleoptiles exhibited Ca2+, and calmodulin-dependent protein phosphorylation in vivo.
通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳和二维凝胶电泳分析了燕麦（ Avena sativa L. ）胚芽鞘切段中的体外和体内蛋白质磷酸化情况。在游离钙离子浓度为1至15微摩尔时，几种多肽的体外磷酸化明显增强。三氟拉嗪、氯丙嗪和萘磺酰胺（W7）显著降低了钙离子刺激的磷酸化。有两种多肽在体外和体内条件下均发生了磷酸化，但其他几种多肽在这两种条件下的磷酸化模式不同，这表明体外磷酸化研究并不能真实反映蛋白质的体内磷酸化模式。三氟拉嗪、W7或乙二醇双（β - 氨基乙醚） - N，N' - 四乙酸（EGTA） + 钙离子载体A23187处理导致对照和生长素处理的胚芽鞘切段体内蛋白质磷酸化水平降低。体内磷酸化后通过二维电泳分析发现某些多肽存在生长素依赖性变化。钙调蛋白拮抗剂对磷酸化的普遍抑制表明，对照和生长素处理的胚芽鞘在体内均表现出钙离子和钙调蛋白依赖性蛋白质磷酸化。
