Murtaugh T J, Gilligan D M, Satir B H
Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York 10461.
J Biol Chem. 1987 Nov 15;262(32):15734-9.
In vivo labeling of Paramecium cells with 32Pi most heavily labels a minor 63-kDa protein that undergoes a rapid, Ca2+-dependent dephosphorylation when the cell is stimulated to release. This stimulus-sensitive phosphoprotein was isolated and purified to apparent homogeneity. A polyclonal affinity purified antibody made against the purified protein recognizes both the phosphorylated and dephosphorylated forms of the protein. The phosphorylated 63-kDa protein is found in the cytosolic fraction; it is slightly acidic with two isoelectric forms at pI 5.8 and 6.2 and probably exists as a monomeric 60-65-kDa polypeptide in the native state. The labeled phosphoamino acid of the protein is phosphoserine. The affinity purified antibody recognizes a third isoelectric form at pI 6.3 that appears unlabeled. The specificity of the antibody was confirmed by showing that it immunoprecipitates the correct protein, i.e. the stimulus-sensitive 63-kDa phosphoprotein. The availability of purified 63-kDa protein as well as an antibody against it will now allow molecular, biochemical, and immunocytochemical studies into the role of this protein in the mechanism of exocytosis.
用³²Pᵢ对草履虫细胞进行体内标记时，标记最强烈的是一种分子量为63 kDa的次要蛋白质，当细胞受到刺激释放时，该蛋白质会经历快速的、依赖Ca²⁺的去磷酸化过程。这种对刺激敏感的磷蛋白被分离并纯化至表观均一。针对纯化蛋白制备的多克隆亲和纯化抗体可识别该蛋白的磷酸化和去磷酸化形式。磷酸化的63 kDa蛋白存在于胞质部分；它呈弱酸性，有两种等电形式，pI分别为5.8和6.2，在天然状态下可能以单体形式的60 - 65 kDa多肽存在。该蛋白的标记磷酸氨基酸是磷酸丝氨酸。亲和纯化抗体识别出一种未被标记的等电形式，其pI为6.3。通过证明抗体能免疫沉淀正确的蛋白，即对刺激敏感的63 kDa磷蛋白，证实了抗体的特异性。纯化的63 kDa蛋白及其抗体的可得性现在将使人们能够对该蛋白在胞吐作用机制中的作用进行分子、生化和免疫细胞化学研究。
