Kinetics of activation of human plasminogen by different activator species at pH 7.4 and 37 degrees C.

Wohl, Robert C.; Summaria, Louis; Robbins, Kenneth C.

doi:10.1016/s0021-9258(19)85983-5

Cited by 196 publications

(68 citation statements)

References 23 publications

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“…The overall architecture of TSV-PA resembles that of trypsin (Figure 1). Although trypsin can also activate plasminogen in vitro [38], TSV-PA can be readily distinguished from trypsin by its high substrate specificity and its insensitivity to soybean trypsin inhibitor [16]. Compared to trypsin, TSV-PA contains a well defined C-terminal extension, which is linked by a disulphide bridge to the main body of the enzyme (Figures 1 and 2).…”

Section: Discussionmentioning

confidence: 99%

“…Compared to trypsin (40% sequence identity) TSV-PA presents four insertions, at positions 36, 95a, 172a and 218, and a sevenresidue C-terminal extension, which is well conserved among snake venom serine proteinases (Figure 3). Unlike tPA and uPA, the basic residues essential for PAI-1 binding are not present in TSV-PA (residues [35][36][37][38][39]. The sequences around the 99-loop, the 60-loop and the 148-loop are also shorter than in tPA and uPA, and a four-residue insertion at position 205 is missing, as in trypsin (Figure 3).…”

Section: Overall Structurementioning

confidence: 99%

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The crystal structure of the novel snake venom plasminogen activator TSV-PA: a prototype structure for snake venom serine proteinases

et al. 1998

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We postulate that Phe 193 accounts for the high substrate specificity of TSV-PA and renders it incapable of forming a stable complex with bovine pancreatic trypsin inhibitor and other extended substrates and inhibitors. Mutational studies previously showed that Asp97 is crucial for the plasminogenolytic activity of TSV-PA, here we identify the conservation of Asp97 in both types of mammalian plasminogen activator - tissue-type (tPA) and urokinase-type (uPA). It seems likely that Asp97 of tPA and uPA will have a similar role in plasminogen recognition. The C-terminal extension of TSV-PA is conserved among snake venom serine proteinases, although its function is unknown. The three-dimensional structure presented here is the first of a snake venom serine proteinase and provides an excellent template for modelling other homologous family members.

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Section: Discussionmentioning

confidence: 99%

Section: Overall Structurementioning

confidence: 99%

The crystal structure of the novel snake venom plasminogen activator TSV-PA: a prototype structure for snake venom serine proteinases

et al. 1998

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“…Steady State Kinetic Parameters of Activation of HPlg by SK Mutants-A one-stage assay as described previously was used to measure HPlg activation by SK mutants (28,29). Briefly, HPlg at final concentrations ranging from 0.04 to 4 M was incubated with 0.5 mM S-2251 in an assay cuvette containing 150 l of 0.05 M Tris buffer, pH 7.4, and 0.1 M NaCl.…”

Section: Methodsmentioning

confidence: 99%

Coiled Coil Region of Streptokinase γ-Domain Is Essential for Plasminogen Activation

Shi

Chuang

et al. 2001

Journal of Biological Chemistry

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The specific functions of the amino acid residues in the streptokinase (SK) gamma-domain were analyzed by studying the interactions of human plasminogen (HPlg) and SK mutants prepared by charge-to-alanine mutagenesis. SK with mutations of groups of amino acids outside the coiled coil region of SK gamma-domain, SK(K278A,K279A,E281A,K282A), and SK(D360A,R363A) had similar HPlg activator activities as wild-type SK. However, significant changes of the functions of SK with mutations within the coiled coil region were observed. Both SK(D322A,R324A,D325A) and SK(R330A,D331A,K332A,K334A) had decreased amounts of complex formation with microplasminogen and failed to activate HPlg. SK(D328A,R330A) had a 21-fold reduced catalytic efficiency for HPlg activation. The studies of SK with single amino acid mutation to Ala demonstrate that Arg(324), Asp(325), Lys(332), and Lys(334) play important roles in the formation of a HPlg.SK complex. On the other hand, amino acid residues Asp(322), Asp(328), and Arg(330) of SK are involved in the virgin enzyme induction. Potential contact between Lys(332) of SK and Glu(623) of human microplasmin and strong interactions between Asp(328) and Lys(330), Asp(331) and Lys(334), and Asp(322) and Lys(334) of SK are noticed. These interactions are important in maintaining a coiled coil conformation. Therefore, we conclude that the coiled coil region of SK gamma-domain, SK(Leu(314)-Ala(342)), plays very important roles in HPlg activation by participating in virgin enzyme induction and stabilizing the activator complex.

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“…For determination of the kinetic parameters of substrate PG activation by activator complex in the presence of SK-peptides, the assay was performed as described above, except that the concentration of peptides used in the assay cuvette was 20 p M , and varying concentrations of either BPG or HPG (0.25-3.0 p M ) were used in the assay buffer. The kinetic parameters for PG activation were calculated by standard methods (Wohl et al, 1980).…”

Section: Assay For Determining the Inhibition Of Activator Activiry Of Sk-hpg Complex By N-terminal Sk-peptidesmentioning

confidence: 99%

Role of the amino‐terminal region of streptokinase in the generation of a fully functional plasminogen activator complex probed with synthetic peptides

et al. 1998

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The mechanism whereby fragments of streptokinase (SK) derived from its N terminus (e.g., SK1-59 or SK1-63) enhance the low plasminogen (PG)-activating ability of other fragments, namely SK64-386, SK60-414, SK60-387, and SK60-333 (reported previously), has been investigated using a synthetic peptide approach. The addition of either natural SK1-59, or chemically synthesized SK16-59, at saturation (about 500-fold molar excess) generated amidolytic and PG activation capabilities in equimolar mixtures of human plasminogen (HPG) and its complementary fragment (either SK60-414 or SK56-414, prepared by expression of truncated SK gene fragments in Escherichia coli) that were approximately 1.2-and 2.5-fold, respectively, of that generated by equimolar mixtures of native SK and HPG. Although in the absence of SK1-59 equimolar mixtures of SK56-414 and HPG could generate almost 80% of amidolytic activity, albeit slowly, less than 2% level of PG activation could be observed under the same conditions, indicating that the contribution of the N-terminal region lay mainly in imparting in SK56-414 an enhanced ability for PG activation. The ability of various synthetic peptides derived from the amino-terminal region (SK16-51, SK16-45, SK37-59, SK1-36, SK16-36, and SK37-51) to (1) complement equimolar mixtures of SK56-414 and HPG for the generation of amidolytic and PG activation functions, (2) inhibit the potentiation of SK56-414 and HPG by SK16-59, and (3) directly inhibit PG activation by the 1:l SK-HPG activator complex was tested. Apart from SK16-59, SK16-51, and 16-45, the ability to rapidly generate amidolytic potential in HPG in the presence of SK56-414 survived even in the smaller SK-peptides, viz., SK37-59 and SK37-51. However, this ability was abolished upon specifically mutating the sequence -LTSRP-, present at position 42-46 in native SK. Although SK16-5 1 retained virtually complete ability for potentiation of PG activation in comparison to SK16-59 or SK1-59, this ability was reduced by approximately fourfold in the case of SK16-45, and completely abolished upon further truncation of the C-terminal residues to SK16-36 or SK1-36. Remarkably, however, these peptides not only displayed ability to bind PG, but also showed strong inhibition of PG activation by the native activator complex in the micromolar range of concentration; the observed inhibition, however, could be competitively relieved by increasing the concentration of substrate PG in the reaction, suggesting that this region in SK contains a site directed specifically toward interaction with substrate PG. This conclusion was substantiated by the observation that the potentiation of PG activating ability was found to be considerably reduced in a peptide (SK25-59) in which the sequence corresponding to this putative locus (residues 16-36) was truncated at the middle. On the other hand, fragments SK37-51 and SK37-59 did not show any inhibition of the PG activation by native activator complex. Taken together, these findings strongly support a model of SK acti...

show abstract

Kinetics of activation of human plasminogen by different activator species at pH 7.4 and 37 degrees C.

Cited by 196 publications

References 23 publications

The crystal structure of the novel snake venom plasminogen activator TSV-PA: a prototype structure for snake venom serine proteinases

The crystal structure of the novel snake venom plasminogen activator TSV-PA: a prototype structure for snake venom serine proteinases

Coiled Coil Region of Streptokinase γ-Domain Is Essential for Plasminogen Activation

Role of the amino‐terminal region of streptokinase in the generation of a fully functional plasminogen activator complex probed with synthetic peptides

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