Kang H M, Kassam G, Jarvis S E, Fitzpatrick S L, Waisman D M
Cell Regulation Research Group, Department of Medical Biochemistry, University of Calgary, Alberta, Canada.
Biochemistry. 1997 Feb 25;36(8):2041-50. doi: 10.1021/bi962569b.
Annexin II tetramer (AIIt) is a Ca2+-dependent, phosphatidylserine-binding, and F-actin-bundling phosphoprotein which is localized to both the extracellular and cytoplasmic surfaces of the plasma membrane. The tetramer is composed of two p36 heavy chains and two p11 light chains. We have produced prokaryotic cDNA expression constructs for both p36 and p11. Both proteins were expressed in large amounts in Escherichia coli upon induction with IPTG. Electrospray ionization mass spectrometry and amino acid sequence analysis of purified recombinant p36 (rp36) and recombinant p11 (rp11) suggested that the recombinant proteins were identical to their native counterparts except for the lack of N-terminal acetylation of rp36. Furthermore, the non-acetylated rp36 bound rp11 and formed AIIt. The circular dichroism spectra and urea denaturation profiles of acetylated AIIt and non-acetylated rAIIt were identical. In addition, both the acetylated AIIt and non-acetylated rAIIt were similar in their Ca2+ dependence and concentration dependence of phospholipid liposome aggregation, chromaffin granule aggregation, and F-actin bundling. These results suggest that N-terminal acetylation of p36 is not in fact necessary for binding of the protein to p11 and that N-terminal acetylation does not affect the conformational stability of AIIt or the in vitro activities of AIIt. The availability of large amounts of rAIIt will facilitate further characterization of the structure-function relationships of the protein.
膜联蛋白II四聚体（AIIt）是一种依赖钙离子、结合磷脂酰丝氨酸且能使F-肌动蛋白成束的磷蛋白，定位于质膜的细胞外表面和细胞质表面。该四聚体由两条p36重链和两条p11轻链组成。我们已经构建了p36和p11的原核cDNA表达载体。用异丙基-β-D-硫代半乳糖苷（IPTG）诱导后，这两种蛋白在大肠杆菌中均大量表达。对纯化的重组p36（rp36）和重组p11（rp11）进行电喷雾电离质谱分析和氨基酸序列分析表明，除rp36缺乏N端乙酰化外，重组蛋白与天然蛋白相同。此外，未乙酰化的rp36与rp11结合并形成AIIt。乙酰化AIIt和未乙酰化rAIIt的圆二色光谱和尿素变性图谱相同。此外，乙酰化AIIt和未乙酰化rAIIt在对钙离子的依赖性以及对磷脂脂质体聚集、嗜铬颗粒聚集和F-肌动蛋白成束的浓度依赖性方面相似。这些结果表明，p36的N端乙酰化实际上并非该蛋白与p11结合所必需，且N端乙酰化不影响AIIt的构象稳定性或AIIt的体外活性。大量rAIIt的可得性将有助于进一步表征该蛋白的结构-功能关系。
