Rajamohan G, Dikshit K L
Division of Molecular Biology, Institute of Microbial Technology, Sector 39A, 160036, Chandigarh, India.
FEBS Lett. 2000 Jun 2;474(2-3):151-8. doi: 10.1016/s0014-5793(00)01578-7.
Staphylokinase (SAK) forms an inactive 1:1 complex with plasminogen (PG), which requires both the conversion of PG to plasmin (Pm) to expose an active site in PG-SAK activator complex and the amino-terminal processing of SAK to expose the positively charged (Lys-11) amino-terminus after removal of the 10 N-terminal amino acid residues from the full length protein. The mechanism by which the N-terminal segment of SAK affects its PG activation capability was investigated by generating SAK mutants, blocked in the native amino-terminal processing site of SAK, and carrying an alteration in the placement of the positively charged amino acid residue, Lys-11, and further studying their interaction with PG, Pm, miniplasmin and kringle structures. A ternary complex formation between PG-SAK PG was observed when an immobilized PG-SAK binary complex interacted with free radiolabelled PG in a sandwich binding experiment. Formation of this ternary complex was inhibited by a lysine analog, 6-aminocaproic acid (EACA), in a concentration dependent manner, suggesting the involvement of lysine binding site(s) in this process. In contrast, EACA did not significantly affect the formation of binary complex formed by native SAK or its mutant derivatives. Furthermore, the binary (activator) complex formed between PG and SAK mutant, PRM3, lacking the N-terminal lysine 11, exhibited 3-4-fold reduced binding with PG, Pm or miniplasmin substrate during ternary complex formation as compared to native SAK. Additionally, activator complex formed with PRM3 failed to activate miniplasminogen and exhibited highly diminished activation of substrate PG. Protein binding studies indicated that it has 3-5-fold reduction in ternary complex formation with miniplasmin but not with the kringle structure. In aggregate, these observations provide experimental evidence for the participation of the N-terminal region of SAK in accession and processing of substrate by the SAK-Pm activator complex to potentiate the PG activation by enhancing and/or stabilizing the interaction of free PG.
葡萄球菌激酶（SAK）与纤溶酶原（PG）形成无活性的1:1复合物，这既需要将PG转化为纤溶酶（Pm）以在PG-SAK激活剂复合物中暴露一个活性位点，也需要对SAK进行氨基末端加工，以便在从全长蛋白中去除10个N端氨基酸残基后暴露带正电荷的（Lys-11）氨基末端。通过生成在SAK天然氨基末端加工位点受阻且带正电荷的氨基酸残基Lys-11位置发生改变的SAK突变体，并进一步研究它们与PG、Pm、微型纤溶酶和kringle结构的相互作用，来探究SAK的N端片段影响其PG激活能力的机制。在夹心结合实验中，当固定化的PG-SAK二元复合物与游离的放射性标记PG相互作用时，观察到了PG-SAK-PG三元复合物的形成。赖氨酸类似物6-氨基己酸（EACA）以浓度依赖的方式抑制了这种三元复合物的形成，表明赖氨酸结合位点参与了这一过程。相比之下，EACA对天然SAK或其突变衍生物形成的二元复合物的形成没有显著影响。此外，与天然SAK相比，在三元复合物形成过程中，PG与缺乏N端赖氨酸11的SAK突变体PRM3形成的二元（激活剂）复合物与PG、Pm或微型纤溶酶底物的结合减少了3-4倍。此外，与PRM3形成的激活剂复合物未能激活微型纤溶酶原，并且对底物PG的激活能力大大降低。蛋白质结合研究表明，它与微型纤溶酶形成三元复合物的能力降低了3-5倍，但与kringle结构形成三元复合物的能力未降低。总的来说，这些观察结果为SAK的N端区域参与SAK-Pm激活剂复合物对底物的结合和加工提供了实验证据，通过增强和/或稳定游离PG的相互作用来增强PG的激活。
