Novokhatny V, Medved L, Lijnen H R, Ingham K
Holland Laboratory, American Red Cross, Rockville, Maryland 20855, USA.
J Biol Chem. 1995 Apr 14;270(15):8680-5. doi: 10.1074/jbc.270.15.8680.
Differential scanning calorimetry was used to study the domain structure and intramolecular interactions of tPA/uPA chimeras. A high temperature transition centered near 90 degrees C was observed upon melting of the tPA/uPA chimera (amino acids 1-274 of tPA and 138-411 of uPA) and its variant lacking the finger and epidermal growth factor-like modules (residues 1-3 and 87-274 of tPA and 138-411 of uPA). Since neither of the two parent plasminogen activators display such a stable structure, one may suggest that a new stabilizing intramolecular interaction occurs in the chimeras. We found that occupation of the lysine binding site of tPA by a lysine or arginine side chain from the urokinase moiety is responsible for the high temperature transition as well as for the failure of the chimeras to exhibit the expected fibrin binding properties. All uPA species, single- and two-chain high molecular weight uPA (Pro-Uk and HMW-Uk) and two-chain low molecular weight uPA (LMW-Uk), interact intermolecularly with tPA and its kringle-containing derivatives. This intermolecular interaction was strongly inhibited by epsilon-aminocaproic acid indicating that the lysine binding site of tPA is involved. The binding of uPA with the fluorescein-labeled A-chain of tPA, registered by changes in fluorescence anisotropy, was estimated to have a Kd range of 1-7 microM. The interaction of tPA with uPA determined by solid-phase assays appeared to be tighter, with a Kd range of 50-300 nM. Two synthetic peptides, with and without carboxyl-terminal lysine, corresponding to urokinase residues 144-158 and 144-157, were approximately 100-fold more potent than epsilon-aminocaproic acid with respect to inhibition of the tPA-uPA interaction, indicating that the tPA binding site on urokinase is located within this sequence, close to the activation site Lys158-Ile159. The discovered intermolecular interaction may be related to the reported synergistic effect of simultaneous administration of these two plasminogen activators.
差示扫描量热法被用于研究组织型纤溶酶原激活剂（tPA）/尿激酶型纤溶酶原激活剂（uPA）嵌合体的结构域结构和分子内相互作用。在tPA/uPA嵌合体（tPA的1 - 274个氨基酸和uPA的138 - 411个氨基酸）及其缺乏指状结构域和表皮生长因子样结构域的变体（tPA的1 - 3、87 - 274个氨基酸和uPA的138 - 411个氨基酸）熔化时，观察到一个以90℃为中心的高温转变。由于两种亲本纤溶酶原激活剂均未表现出这种稳定结构，因此可以推测嵌合体中发生了一种新的稳定分子内相互作用。我们发现，来自尿激酶部分的赖氨酸或精氨酸侧链占据tPA的赖氨酸结合位点，这是高温转变以及嵌合体未能表现出预期纤维蛋白结合特性的原因。所有uPA种类，单链和双链高分子量uPA（单链尿激酶原和高分子量尿激酶）以及双链低分子量uPA（低分子量尿激酶），均与tPA及其含kringle结构域的衍生物发生分子间相互作用。这种分子间相互作用被ε-氨基己酸强烈抑制，表明tPA的赖氨酸结合位点参与其中。通过荧光偏振变化记录的uPA与荧光素标记的tPA A链的结合，估计其解离常数（Kd）范围为1 - 7微摩尔。通过固相分析测定的tPA与uPA的相互作用似乎更强，Kd范围为50 - 300纳摩尔。两种合成肽，一种含有羧基末端赖氨酸，另一种不含，对应于尿激酶的144 - 158和144 - 157位残基，在抑制tPA - uPA相互作用方面比ε-氨基己酸强约100倍，这表明尿激酶上的tPA结合位点位于该序列内，靠近激活位点Lys158 - Ile159。所发现的分子间相互作用可能与报道的同时给予这两种纤溶酶原激活剂的协同效应有关。
