Harris H W, Lux S E
J Biol Chem. 1980 Dec 10;255(23):11512-20.
The phosphorylation sites of spectrin dimer were characterized before and after incubation of intact human red cells with [32P]orthophosphate. Phosphate measurements of unlabeled spectrin dimer show it contains 4.0 +/- 0.3 covalent protein phosphates, all located on band 2. Quantitation of the number of exchangeable phosphorylation sites in erythrocytes labeled with [32P]orthophosphate yields 3.9 +/- 0.3 phosphates/spectrin dimer, indicating that all four spectrin phosphorylation sites are metabolically active. Tryptic and chymotryptic peptide mapping reveals that the dimer contains three unique 32P-labeled tryptic peptides of approximately 4,600 (A1), 3,500 (A2), and 2,400 (B) daltons. Peptide A1 contains both phosphoserine and phosphothreonine while peptides A2 and B possess only phosphoserine. Peptides A1 and A2 remain associated after trypsinization of spectrin dimer and are only separable in detergents. All three 32P-labeled tryptic peptides are contained within a 20,000 dalton cyanogen bromide fragment which is within a 60,000 dalton staphylococcal protease phosphopeptide. All these fragments are found within a 90,000 dalton nitrothiocyanobenzoic acid phosphopeptide. The purified 20,000 dalton fragment contains no homoserine or homoserine lactone and is the COOH-terminal cyanogen bromide peptide of band 2. The isolated tryptic peptide B possesses no lysine or arginine and is presumably the COOH-terminal tryptic peptide of band 2 and the most distal phosphopeptide. Thus, the four phosphorylation sites of spectrin dimer are clustered at the extreme COOH-terminal end of band 2.
在用[32P]正磷酸盐孵育完整的人红细胞之前和之后，对血影蛋白二聚体的磷酸化位点进行了表征。未标记的血影蛋白二聚体的磷酸盐测量显示，它含有4.0±0.3个共价蛋白磷酸盐，均位于带2上。对用[32P]正磷酸盐标记的红细胞中可交换磷酸化位点数量的定量分析得出，每个血影蛋白二聚体有3.9±0.3个磷酸盐，这表明所有四个血影蛋白磷酸化位点都具有代谢活性。胰蛋白酶和糜蛋白酶肽图谱分析表明，该二聚体含有三个独特的32P标记的胰蛋白酶肽，分子量约为4600（A1）、3500（A2）和2400（B）道尔顿。肽A1同时含有磷酸丝氨酸和磷酸苏氨酸，而肽A2和B仅含有磷酸丝氨酸。血影蛋白二聚体经胰蛋白酶消化后，肽A1和A2仍结合在一起，只有在去污剂中才能分离。所有三个32P标记的胰蛋白酶肽都包含在一个20000道尔顿的溴化氰片段中，该片段位于一个60000道尔顿的葡萄球菌蛋白酶磷酸肽内。所有这些片段都存在于一个90000道尔顿的硝基硫氰基苯甲酸磷酸肽中。纯化的20000道尔顿片段不含高丝氨酸或高丝氨酸内酯，是带2的羧基末端溴化氰肽。分离出的胰蛋白酶肽B不含赖氨酸或精氨酸，推测是带2的羧基末端胰蛋白酶肽和最远端的磷酸肽。因此，血影蛋白二聚体的四个磷酸化位点聚集在带2的极端羧基末端。
