Regnouf F, Rendon A, Pradel L A
Laboratoire de Biophysique, Institut de Biologie Physico-Chimique, Paris, France.
J Neurochem. 1991 Jun;56(6):1985-96. doi: 10.1111/j.1471-4159.1991.tb03457.x.
Five proteins having molecular masses of 90, 67, 37, 36, and 32 kDa (p90, p67, p37, p36, and p32, respectively) were identified in the particulate fractions of pig brain cortex and pig spinal cord prepared in the presence of 0.2 mM Ca2+ and further purified using a protocol previously described for the purification of calpactins. Proteins p90, p37, and p36 are related to annexins I and II. Annexin II, represented by p90, is found as an heterotetramer, composed of two heavy chains of 36 kDa and two light chains of 11 kDa, and as a monomer of 36 kDa. Protein p37, which differs immunologically from p36, is a monomer and could be related to annexin I. All three proteins are Ca(2+)-dependent phospholipid- and F-actin-binding proteins; they are phosphorylated on a serine and on a tyrosine residue by protein kinases associated with synaptic plasma membranes. Purified p36 monomer and p36 heterotetramer proteins bind to actin at millimolar Ca2+ concentrations. The stoichiometry of p36 binding to F-actin at saturation is 1:2, corresponding to one tetramer or monomer of calpactin for two actin monomers (KD, 3 x 10(-6) M). Synaptic plasma membranes supplemented with the monomeric or tetrameric forms of p36 phosphorylate the proteins on a serine residue. The monomer is phosphorylated on a serine residue by a Ca(2+)-independent protein kinase, whereas the heterotetramer is phosphorylated on a serine residue and a tyrosine residue by Ca(2+)-dependent protein kinases. Antibodies to brain p37 and p36 together with antibodies to lymphocytes lipocortins 1 and 2 were used to follow the distribution of these proteins in nervous tissues. Polypeptides of 37, 34, and 36 kDa cross-react with these antibodies. Anti-p37 and antilipocortin 1 cross-react on the same 37- and 34-kDa polypeptides; anti-p36 and antilipocortin 2 cross-react only on the 36-kDa polypeptides.
在存在0.2 mM Ca2+的条件下制备的猪脑皮层和猪脊髓的颗粒组分中，鉴定出了分子量分别为90、67、37、36和32 kDa的五种蛋白质（分别为p90、p67、p37、p36和p32），并使用先前描述的钙结合蛋白纯化方案进一步纯化。蛋白质p90、p37和p36与膜联蛋白I和II相关。以p90为代表的膜联蛋白II以异源四聚体形式存在，由两条36 kDa的重链和两条11 kDa的轻链组成，也以36 kDa的单体形式存在。在免疫学上与p36不同的蛋白质p37是一种单体，可能与膜联蛋白I相关。所有这三种蛋白质都是Ca(2+)依赖性磷脂和F-肌动蛋白结合蛋白；它们在与突触质膜相关的蛋白激酶作用下，在丝氨酸和酪氨酸残基上发生磷酸化。纯化的p36单体和p36异源四聚体蛋白在毫摩尔浓度的Ca2+条件下与肌动蛋白结合。饱和时p36与F-肌动蛋白结合的化学计量比为1:2，对应于两个肌动蛋白单体结合一个钙结合蛋白四聚体或单体（KD，3×10(-6) M）。补充有p36单体或四聚体形式的突触质膜在丝氨酸残基上使蛋白质磷酸化。单体在一个丝氨酸残基上被一种不依赖Ca(2+)的蛋白激酶磷酸化，而异源四聚体在一个丝氨酸残基和一个酪氨酸残基上被依赖Ca(2+)的蛋白激酶磷酸化。使用针对脑p37和p36的抗体以及针对淋巴细胞脂皮质素1和2的抗体来追踪这些蛋白质在神经组织中的分布。37、34和36 kDa的多肽与这些抗体发生交叉反应。抗p37和抗脂皮质素1在相同的37和34 kDa多肽上发生交叉反应；抗p36和抗脂皮质素2仅在36 kDa多肽上发生交叉反应。
