| Summary: | 博士 === 國立成功大學 === 基礎醫學研究所 === 86 === Streptokinase (SK), a single-peptide secretory protein of 414
amino acid residues, is produced from various strains of
b-hemolytic Streptococci in Lancefield group A, C and G. SK
can activate human plasminogen (HPlg) to human plasmin (HPlm) potently, therefore, it is used as a thrombolytic agent to dissolve thromboembolic clots in blood vessels, such as acute myocardial infarction. SK itself does not have proteolytic activity. The activation of HPlg by SK-type activator may be via a unique mechanism involving the modulation of HPlg molecule. The role of SK moiety in HPlg activation has been proposed to have two components: (a) SK may open the active center on one HPlg, thereby inducing it to serve as an activator HPlg; (b) SK may modify the geometry near the Arg560-Val561 bond of the substrate HPlg for an efficient cleavage. In this study, the goals aimed to clarify more precisely the mechaism of HPlg activation by SK, in searching of the functions of various domains of SK and the interaction modes between SK and HPlg.
A series of recombinant truncated SK peptides with deletion of
NH2 and/or COOH-terminal amino acid residues were prepared by
gene expression and protein purification from Escherichia coli
system. In assaying of the enzymatic activities, SK(16-414)
and SK(16-378) were as acitve as native SK in HPlg activation.
SK(60-414), with the modification by SK(1-59), could also
serve as a HPlg activator. SK(91-414), SK(127-414) and SK(158-414),
at catalytic concentrations, all failed to activate HPlg.
However, these truncated SK peptides retained the ability to
expose amidolytically "virgin enzyme"-active center in a 1:1
stoichiometric complex with HPlg. SK(16-316) could neither
transform HPlg to "virgin enzyme", nor catalyze the HPlg
activation, but the HPlg moiety was slowly converted to HPlm
in equimolar HPlg-SK(16-316) complex. Although SK(16-251) and
SK(220-414) alone didn''t have any enzymatic activity,
combination of SK(16-251) and SK(220-414) allowed the two
peptides in complement with each other to form an efficient
HPlg activator. In addition, SK(16-251) dose-dependently
enhanced the activation of HPlg by SK(16-414), while the HPlg
activation by SK(16-414) was inhibited by SK(220-414). Based
on the concentration-dependent binding assay and competitive
binding assay, three interaction modes between SK and HPlg
domains were demonstrated as follows: SK(220-414) and catalytic
domain of HPlg, SK(16-251) and HPlg kringle domain as well as
SK(220-414) and HPlg kringle domain. Furthermore, SK(16-251)
and SK(220-414) could compete for binding on the same or nearby
locations on HPlg kringle domain. In the sandwich-binding
assay, a HPlg-specific ternary complex in form of HPlg-SK-HPlg
was detected. Formation of the ternary complex may represent
an enzyme-substrate intermediate for HPlg activation.
In conclusion, one SK can interact with two HPlg molecules
simultaneously. The C-terminal domain of SK is responsible
for the formation of enzymatic center in complexing with HPlg,
whereas the NH2-terminal domain of SK acts as a substrate
modulator. As bridged with SK, the HPlg that binds to the
C-terminal domains of SK functions as an enzyme to efficiently
catalyze the conversion of substrate HPlg that binds to the
NH2-terminal domain of SK to human plasmin.
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