Langen H T, Taylor J W
Laboratory of Bioorganic Chemistry and Biochemistry, Rockefeller University, New York, New York 10021.
Proteins. 1992 Sep;14(1):1-9. doi: 10.1002/prot.340140103.
By inserting appropriate peptide ligands into surface loops on globular proteins, we expect to develop probes for the location, accessibility, and steric and electrostatic environment of these ligand-binding sites on their membrane-bound receptors. Three residues in a loop on the surface of E. coli alkaline phosphatase were substituted by an 18-residue peptide containing the receptor-binding segment of somatostatin-14 without significantly affecting the catalytic properties of the enzyme. This hybrid protein was then used to investigate the ligand-binding site of somatostatin receptors. Tryptic cleavage of the hybrid protein within the inserted sequence, and binding of the hybrid protein to antisomatostatin antibodies demonstrated the surface accessibility of the guest peptide. Both the wild-type enzyme and the hormone-enzyme hybrid displaced 125I-labeled somatostatin from rat brain membrane receptors only at high concentrations. However, chemical cationization of the hybrid protein, which again did not disturb the phosphatase activity, enhanced its receptor-binding potency to a level only 23 times lower than that of somatostatin itself and 280 times higher than that of the cationized wild-type protein. This alkaline phosphatase/somatostatin hybrid protein appears, therefore, to be a suitable starting point for the development of probes for the steric and electrostatic environment of the ligand-binding site of somatostatin receptors.
通过将合适的肽配体插入球状蛋白的表面环中，我们期望开发出用于研究这些配体结合位点在其膜结合受体上的位置、可及性以及空间和静电环境的探针。大肠杆菌碱性磷酸酶表面环中的三个残基被一个含有生长抑素-14受体结合片段的18个残基的肽取代，而对该酶的催化特性没有显著影响。然后利用这种杂合蛋白来研究生长抑素受体的配体结合位点。在插入序列内对杂合蛋白进行胰蛋白酶切割，以及杂合蛋白与抗生长抑素抗体的结合，证明了客体肽的表面可及性。野生型酶和激素-酶杂合蛋白都只有在高浓度时才能从大鼠脑膜受体上置换出125I标记的生长抑素。然而，杂合蛋白的化学阳离子化（这同样不会干扰磷酸酶活性）将其受体结合能力提高到仅比生长抑素本身低23倍、比阳离子化野生型蛋白高280倍的水平。因此，这种碱性磷酸酶/生长抑素杂合蛋白似乎是开发用于研究生长抑素受体配体结合位点的空间和静电环境的探针的合适起点。
