Nakagawa S H, Tager H S
J Biol Chem. 1986 Jun 5;261(16):7332-41.
To gain an understanding of the causes of decreased biological activity in insulins bearing amino acid substitutions at position B25 and the importance of the PheB25 side chain in directing hormone-receptor interactions, we have prepared a variety of insulin analogs and have studied both their interactions with isolated canine hepatocytes and their abilities to stimulate glucose oxidation by isolated rat adipocytes. The semisynthetic analogs fall into three structural classes: (a) analogs in which the COOH-terminal 5, 6, or 7 residues of the insulin B-chain have been deleted, but in which the COOH-terminal residue of the B-chain has been derivatized by alpha-carboxamidation; (b) analogs in which PheB25 has been replaced by unnatural aromatic or natural L-amino acids; and (c) analogs in which the COOH-terminal 5 residues of the insulin B-chain have been deleted and in which residue B25 has been replaced by selected alpha-carboxamidated amino acids. Our results showed that (a) insulin residues B26-B30 can be deleted without decrease in biological potency, whereas deletion of residues B25-B30 and B24-B30 causes a marked and cumulative decrease in potency; (b) replacement of PheB25 in insulin by Leu or Ser results in analogs with biological potency even less than that observed when residues B25-B30 are deleted; (c) the side chain bulk of naphthyl(1)-alanine or naphthyl(2)-alanine at position B25 is well tolerated during insulin interactions with receptor, whereas that of homophenylalanine is not; and (d) the decreased biological potency attending substitution of insulin PheB25 by Ala, Ser, Leu, or homophenylalanine is reversed, in part or in total, by deletion of COOH-terminal residues B26-B30. Additional experiments showed that the rate of dissociation of receptor-bound 125I-labeled insulin from isolated hepatocytes is enhanced by incubating cells with insulin or [naphthyl(2)-alanineB25]insulin, but not with analogs in which PheB25 is replaced by serine, leucine, or homophenylalanine; deletion of residues B26-B30, however, results in analogs that enhance the rate of dissociation of receptor-bound insulin in all cases studied. We conclude that (a) steric hindrance involving the COOH-terminal domain of the B chain plays a major role in directing the interaction of insulin with its receptor; (b) the initial negative effect of this domain is reversed upon the filling of a site reflecting interaction of the receptor and the beta-aromatic ring of the PheB25 side chain.(ABSTRACT TRUNCATED AT 400 WORDS)
为了了解在B25位带有氨基酸取代的胰岛素生物活性降低的原因以及PheB25侧链在指导激素-受体相互作用中的重要性，我们制备了多种胰岛素类似物，并研究了它们与分离的犬肝细胞的相互作用以及刺激分离的大鼠脂肪细胞葡萄糖氧化的能力。这些半合成类似物分为三类结构：（a）胰岛素B链的羧基末端5、6或7个残基被删除，但B链的羧基末端残基通过α-羧酰胺化进行了衍生化；（b）PheB25被非天然芳香族或天然L-氨基酸取代的类似物；（c）胰岛素B链的羧基末端5个残基被删除且B25残基被选定的α-羧酰胺化氨基酸取代的类似物。我们的结果表明：（a）胰岛素残基B26-B30可以被删除而不降低生物活性，而删除残基B25-B30和B24-B30会导致活性显著且累积性降低；（b）用Leu或Ser取代胰岛素中的PheB25会产生生物活性甚至低于删除B25-B30残基时所观察到的类似物；（c）在胰岛素与受体相互作用期间，B25位萘基（1）-丙氨酸或萘基（2）-丙氨酸的侧链体积是可以被很好耐受的，而高苯丙氨酸的则不行；（d）用Ala、Ser、Leu或高苯丙氨酸取代胰岛素PheB25导致的生物活性降低部分或全部通过删除羧基末端残基B26-B30而得到逆转。额外的实验表明，通过用胰岛素或[萘基（2）-丙氨酸B25]胰岛素孵育细胞，可增强受体结合的125I标记胰岛素从分离的肝细胞上的解离速率，但用PheB25被丝氨酸、亮氨酸或高苯丙氨酸取代的类似物则不行；然而，在所有研究的情况下，删除残基B26-B30会产生能增强受体结合胰岛素解离速率的类似物。我们得出结论：（a）涉及B链羧基末端结构域的空间位阻在指导胰岛素与其受体的相互作用中起主要作用；（b）该结构域的初始负面影响在反映受体与PheB25侧链β-芳香环相互作用的位点被填满后会被逆转。（摘要截取自400词）
