scholarly journals Function of streptokinase fragments in plasminogen activation

1994 ◽  
Vol 304 (1) ◽  
pp. 235-241 ◽  
Author(s):  
G Y Shi ◽  
B I Chang ◽  
S M Chen ◽  
D H Wu ◽  
H L Wu
Keyword(s):  
Amino Acid ◽  
Plasminogen Activation ◽  
Peptide Fragment ◽  
Peptide Fragments ◽  
Reverse Phase ◽  
Amino Acid Peptide ◽  
High Affinity ◽  
Catalytic Constant ◽  
Molecular Masses ◽  
Human Plasminogen

Several peptide fragments of streptokinase (SK) were prepared by incubating SK with immobilized human plasmin (hPlm) and purified by h.p.l.c. with a reverse-phase phenyl column. The N-terminal sequences, amino acid compositions and molecular masses of these peptide fragments were determined. The SK peptide fragment of 36 kDa consisting of Ser60-Lys387 (SK-p), was the only peptide fragment that could be tightly bound to immobilized hPlm. Another three large SK peptide fragments, SK-m, SK-n and SK-o, with molecular masses of 7 kDa, 18 kDa and 30 kDa, and consisting of Ile1-Lys59, Glu148-Lys333, Ser60-Lys333 respectively, were also obtained from the supernatant of the reaction mixture. The purified SK-p had high affinity with hPlm and could activate human plasminogen (hPlg) with a kPlg one-sixth that of the native SK. SK-o had low affinity with hPlm and could also activate hPlg, although the catalytic constant was less than 1% of the native SK. SK-n, as well as SK-m, which is the N-terminal 59 amino acid peptide of the native SK, had no activator activity. However, SK-m could enhance the activator activity of both SK-o and SK-p and increase their second-order rate constants by two- and six-fold respectively. It was concluded from these studies that (1) SK-o, the Ser60-Lys333 peptide of SK, was essential for minimal SK activator activity, (2) the C-terminal peptide of SK-p, Ala334-Lys387, was essential for high affinity with hPlm, and (3) the N-terminal 59-amino-acid peptide was important in maintaining the proper conformation of SK to have its full activator activity.

Preparation of a Novel Streptokinase Mutant with Improved Stability

1998 ◽  
Vol 79 (05) ◽  
pp. 992-997 ◽  
Author(s):  
Guey-Yueh Shi ◽  
Bi-Ing Chang ◽  
Su-Wen Su ◽  
Kung-Chia Young ◽  
Dung-Ho Wu ◽  
...  
Keyword(s):  
Half Life ◽  
The Other ◽  
The Novel ◽  
Peptide Fragment ◽  
Peptide Fragments ◽  
Original Activity ◽  
Peptide Bonds ◽  
Improved Stability ◽  
Molecular Masses ◽  
Human Plasminogen

SummaryThe novel mutant streptokinase, SK-K59E, can activate human plasminogen as efficiently as the purified commercially available streptokinase. Several peptide bonds including Lys59-Ser60 in native streptokinase were hydrolyzed in reaction with plasmin and peptides of small molecular masses were generated. The plasminogen activator activity of native streptokinase in reaction with human plasmin declined to 25% of the original activity in a 120-min incubation. On the other hand, the NH2-terminal peptide of SK-K59E remained intact in reaction with plasmin and the activator activity of streptokinase decreased to 75% of the original activity in 120 min. The major degraded peptide fragments of native streptokinase in reaction with plasmin had molecular masses of 36 and 30 kDa. However, two major peptide fragments of 42 and 34 kDa were observed in the reaction of SK-K59E with human plasmin. The 42 kDa peptide fragment, which contained NH2-terminal of streptokinase, could activate human plasminogen as efficiently as the native streptokinase. SK-K59E can induce greater degree of caseinolysis and fibrinolysis than the native streptokinase. In conclusion, the results demonstrate that the prevention of cleavage at Lys59 of streptokinase prolongs the half-life of streptokinase in complex with plasmin and that the NH2-terminal of streptokinase (Ile1-Lys59) plays an important role in maintaining its stability.

Molecular Conversions of Recombinant Staphylokinase During Plasminogen Activation in Purified Systems and in Human Plasma

1993 ◽  
Vol 70 (03) ◽  
pp. 495-499 ◽  
Author(s):  
S Ueshima ◽  
K Silence ◽  
D Collen ◽  
H R Lijnen
Keyword(s):  
Amino Acid ◽  
Human Plasma ◽  
Catalytic Amount ◽  
Clot Lysis ◽  
Lag Phase ◽  
Plasminogen Activation ◽  
Plasma Clot ◽  
Acid Protein ◽  
Human Plasminogen

SummaryRecombinant staphylokinase (STAR) is produced as a 136 amino acid protein with NH2-terminal sequence Ser-Ser-Ser (mature STAR, HMW-STAR), which may be converted to lower molecular weight forms (LMW-STAR) by removal of the first six residues (yielding STAR-Δ6 with NH2-terminal Gly-Lys-Tyr-) or the first ten residues (yielding STAR-Δ10 with NH2-terminal Lys-Gly-Asp-). In the present study the occurrence and effects of these conversions during plasminogen activation by HMW-STAR were studied in purified systems and in human plasma.In stoichiometric mixtures of HMW-STAR and native human plasminogen (Glu-plasminogen), rapid and quantitative conversion of HMW-STAR to LMW-STAR occurred, concomitant with exposure of the active site in the plasmin-STAR complex. NH2-terminal amino acid sequence analysis revealed the sequence Lys-Gly-Asp- in addition to the known sequences of the Lys-plasmin chains, identifying STAR-Δ10 as the derivative generated from HMW-STAR. In mixtures of catalytic amount of HMW-STAR and human plasminogen, plasmin generation occurred progressively, following an initial lag phase, during which HMW-STAR was converted to LMW-STAR. Plasmin-mediated conversion of HMW-STAR to LMW-STAR obeyed Michaelis-Menten kinetics with K m = 3.6 μM and k 2 = 0.38 s−1. The specific clot lysis activities of HMW-STAR (122,000 ± 8,000 units/mg) and LMW-STAR (129,000 ± 8,000 units/mg) were indistinguishable.In an in vitro system consisting of a 60 μl plasma clot submerged in 250 μl plasma, 80% clot lysis within 1 h was obtained with 70 nM HMW-STAR. This was associated with fibrinogen depletion and conversion of 20% of the HMW-STAR to LMW-STAR. Addition of 100 nM HMW-STAR to human plasma in the absence of a clot did not induce significant fibrinogen breakdown (≥ 90% residual fibrinogen after 2 h), and was not associated with significant coversion to LMW-STAR (≤10% within 2 h). With 400 nM HMW-STAR, fibrinogen depletion in plasma occurred within 1 h, and 80% conversion to LMW-STAR was observed. Thus, at fibrinolytically active concentrations which do not cause fibrinogen breakdown, no significant conversion of HMW-STAR to LMW-STAR occurs in human plasma in the absence of a clot.These findings indicate that the conversion of HMW-STAR to LMW-STAR may occur in association with clot lysis, but is not required to induce it.

A 28-Amino-Acid Peptide Fragment of the Cupredoxin Azurin Prevents Carcinogen-Induced Mouse Mammary Lesions

2010 ◽  
Vol 3 (10) ◽  
pp. 1351-1360 ◽  
Author(s):  
Rajeshwari R. Mehta ◽  
Michael Hawthorne ◽  
Xinjian Peng ◽  
Ann Shilkaitis ◽  
Rajendra G. Mehta ◽  
...  
Keyword(s):  
Amino Acid ◽  
Peptide Fragment ◽  
Amino Acid Peptide

A 22-amino-acid peptide restores DNA-binding activity to dimerization-defective mutants of the estrogen receptor

1990 ◽  
Vol 10 (10) ◽  
pp. 5529-5531
Author(s):  
J A Lees ◽  
S E Fawell ◽  
R White ◽  
M G Parker
Keyword(s):  
Estrogen Receptor ◽  
Amino Acid ◽  
Dna Binding ◽  
Binding Activity ◽  
Binding Domain ◽  
Amino Acid Peptide ◽  
High Affinity ◽  
Dna Binding Activity ◽  
Point Mutagenesis ◽  
Hormone Binding Domain

We have identified residues within the estrogen receptor that are required for dimerization and high-affinity DNA binding. A 22-amino-acid peptide encompassing these residues was sufficient to restore DNA-binding activity to a mutant receptor lacking most of the hormone-binding domain. Point mutagenesis of the fusion protein confirmed that this sequence continued to mediate dimerization in a manner similar to that within the native receptor, although its position relative to the DNA-binding domain was appreciably altered.

scholarly journals A 22-amino-acid peptide restores DNA-binding activity to dimerization-defective mutants of the estrogen receptor.

1990 ◽  
Vol 10 (10) ◽  
pp. 5529-5531 ◽  
Author(s):  
J A Lees ◽  
S E Fawell ◽  
R White ◽  
M G Parker
Keyword(s):  
Estrogen Receptor ◽  
Amino Acid ◽  
Dna Binding ◽  
Binding Activity ◽  
Binding Domain ◽  
Amino Acid Peptide ◽  
High Affinity ◽  
Dna Binding Activity ◽  
Point Mutagenesis ◽  
Hormone Binding Domain

We have identified residues within the estrogen receptor that are required for dimerization and high-affinity DNA binding. A 22-amino-acid peptide encompassing these residues was sufficient to restore DNA-binding activity to a mutant receptor lacking most of the hormone-binding domain. Point mutagenesis of the fusion protein confirmed that this sequence continued to mediate dimerization in a manner similar to that within the native receptor, although its position relative to the DNA-binding domain was appreciably altered.

scholarly journals Purification and partial characterization of a novel lectin from elder (Sambucus nigra L.) fruit

1991 ◽  
Vol 278 (3) ◽  
pp. 667-671 ◽  
Author(s):  
L Mach ◽  
W Scherf ◽  
M Ammann ◽  
J Poetsch ◽  
W Bertsch ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Amino Acid ◽  
Carbohydrate Specificity ◽  
Sambucus Nigra ◽  
Acid Glycoprotein ◽  
High Affinity ◽  
Sambucus Nigra Agglutinin ◽  
Sds Page ◽  
Molecular Masses

A previously unknown haemagglutinin, named Sambucus nigra agglutinin-III (SNA-III), has been purified from the fruit of the elder (Sambucus nigra). Whereas elder bark agglutinin I (SNA-I) is highly specific for terminal alpha 2,6-linked sialic acid residues, SNA-III displays a high affinity for oligosaccharides containing exposed N-acetylgalactosamine and galactose residues. Different N-terminal sequences and the amino acid composition distinguish the fruit lectin from elder bark agglutinin II (SNA-II), which shows a similar carbohydrate specificity. The 40-fold higher affinity of SNA-III for asialofetuin than for human asialo-alpha 1-acid glycoprotein and human asialotransferrin respectively suggests a preference for O-linked glycans. SNA-III occurs mainly as a monomeric glycoprotein, but tends to form di- and oligo-meric aggregates. This aggregation seems to mediate the multivalent interaction, leading to agglutination. SDS/PAGE revealed two major polypeptides with apparent molecular masses of 32 and 33 kDa respectively. This heterogeneity is probably a result of proteolysis in the C-terminal region. Binding to concanavalin A and susceptibility to peptide: N-glycosidase F indicated the presence of N-glycosidically linked oligosaccharides.

scholarly journals A cellular binding site for the Mr 55,000 form of the human plasminogen activator, urokinase.

1985 ◽  
Vol 100 (1) ◽  
pp. 86-92 ◽  
Author(s):  
J D Vassalli ◽  
D Baccino ◽  
D Belin
Keyword(s):  
Cell Migration ◽  
Plasminogen Activator ◽  
Plasminogen Activators ◽  
Plasminogen Activation ◽  
Blood Monocytes ◽  
High Affinity ◽  
Human Plasminogen ◽  
A Chain ◽  
Optimal Site ◽  
Extracellular Proteolysis

The secretion of plasminogen activators has been implicated in the controlled extracellular proteolysis that accompanies cell migration and tissue remodeling. We found that the human plasminogen activator urokinase (Uk) (Mr 55,000 form) binds rapidly, specifically, and with high affinity to fresh human blood monocytes and to cells of the monocyte line U937. Upon binding Mr 55,000 Uk was observed to confer high plasminogen activator activity to the cells. Binding of the enzyme did not require a functional catalytic site (located on the B chain of the protein) but did require the noncatalytic A chain of Mr 55,000 Uk, since Mr 33,000 Uk did not bind. These results demonstrate the presence of a membrane receptor for Uk on monocytes and show a hitherto unknown function for the A chain of Uk: binding of secreted enzyme to its receptor results in Uk acting as a membrane protease. This localizes plasminogen activation near the cell surface, an optimal site to facilitate cell migration.

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