Sequences of antigenic epitopes of streptokinase identified via random peptide libraries displayed on phage 1 1Edited by J. A. Wells

Parhami-Seren, Behnaz; Keel, Tracy; Reed, Guy L.

doi:10.1006/jmbi.1997.1174

Cited by 21 publications

(12 citation statements)

References 46 publications

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“…The wells were washed and blocked with 1% BSA before the addition of 50 l of rSK1-59 (native or mutant, 0 -100 g/ml). After a 1-h incubation and washing, the anti-SK NH 2 -terminal mAb 9D10 (23,24) was added for 1 h. After washing, bound mAb was detected by 125 I goat anti-mouse Ab (50,000 cpm) followed by ␥-counting.…”

Section: Discussionmentioning

confidence: 99%

“…After that rSK1-59 (native or mutant, 50 l, 0 -100 g/ml) was added. After a 1-h incubation and washing, the anti-SK NH 2 -terminal monoclonal antibody (mAb) 9D10 (23,24) was added for 1 h. After washing bound mAb was detected by 125 I goat anti-mouse Ab (50,000 cpm) followed by ␥-counting. Binding of rSK1-59 and rSK⌬59 to Glu-Pg-Microtiter plates were coated with 50 l of Glu-Pg (5 g/ml) or no protein (control).…”

Section: Binding Assaysmentioning

confidence: 99%

“…6). To detect whether the L42A mutation altered the structure of the A domain when it interacted with rSK⌬59, we used a panel of well characterized mAbs that bind to conformation-dependent and -independent epitopes in native SK (23,24). The mAbs that recognize the native conformation of the A domain of SK (1E10 and 2E8, Fig.…”

Section: Leu-42 Of Streptokinase Is Critical For Plasminogen Activationmentioning

confidence: 99%

“…For example, a monoclonal antibody against the NH 2 terminus of SK inhibited Pg activation but not amidolytic activity (23,24). The fibronectin motif sequence L 42 TSRPA 47 has been implicated in Pg binding because of its homology to a fragment of fibronectin that binds to plasmin (13).…”

Section: Figmentioning

confidence: 99%

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Leucine 42 in the Fibronectin Motif of Streptokinase Plays a Critical Role in Fibrin-independent Plasminogen Activation

Liu

Sazonova

Turner

et al. 2000

Journal of Biological Chemistry

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The NH 2 terminus (residues 1-59) of streptokinase (SK) is a molecular switch that permits fibrin-independent plasminogen activation. Targeted mutations were made in recombinant (r) SK1-59 to identify structural interactions required for this process. Mutagenesis established the functional roles of Phe-37and Glu-39, which were projected to interact with microplasmin in the activator complex. Mutation of Leu-42 (rSK1-59 L42A ), a conserved residue in the SK fibronectin motif that lacks interactions with microplasmin, strongly reduced plasminogen activation (k cat decreased 50-fold) but not amidolysis (k cat decreased 1.5-fold). Otherwise rSK1-59 L42A and native rSK1-59 were indistinguishable in several parameters. Both displayed saturable and specific binding to Glu-plasminogen or the remaining SK fragment (rSK⌬59). Similarly rSK1-59 and rSK1-59 L42A bound simultaneously to two different plasminogen molecules, indicating that both plasminogen binding sites were intact. However, when bound to SK⌬59, rSK1-59 L42A was less effective than rSK1-59 in restructuring the native conformation of the SK A domain, as detected by conformation-dependent monoclonal antibodies. In the light of previous studies, these data provide evidence that SK1-59 contributes to fibrin-independent plasminogen activation through 1) intermolecular interactions with the plasmin in the activator complex, 2) binding interactions with the plasminogen substrate, and 3) intramolecular interactions that structure the A domain of SK for Pg substrate processing.

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Section: Discussionmentioning

confidence: 99%

Section: Binding Assaysmentioning

confidence: 99%

Section: Leu-42 Of Streptokinase Is Critical For Plasminogen Activationmentioning

confidence: 99%

Section: Figmentioning

confidence: 99%

See 2 more Smart Citations

Leucine 42 in the Fibronectin Motif of Streptokinase Plays a Critical Role in Fibrin-independent Plasminogen Activation

Liu

Sazonova

Turner

et al. 2000

Journal of Biological Chemistry

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“…Sepharose 4B (Amersham Pharmacia) was activated as described. 19 Five to ten milligrams of antibody (Ab) was coupled to 1 mL of activated Sepharose (Ͼ90% of mAb bound). This mAb was selected for studies because of the high yields in ascites and lack of aggregation.…”

Section: Methodsmentioning

confidence: 99%

Ouabain-Binding Protein(s) From Human Plasma

et al. 2002

Self Cite

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Abstract-Conservation of the binding site on mammalian Na ϩ ,K ϩ -ATPase for cardiac glycosides and the importance of the Na ϩ pump in mammalian cellular physiology has stimulated the search for a mammalian analog of these plant compounds. One candidate, isolated from brain and blood, appears to be ouabain itself or a closely related isomer, the ouabain-like compound. Little is known about the circulating form. Because human steroid hormones circulate with carrier proteins, we produced a ouabain-specific monoclonal antibody (mAb 1-10) and used it to probe normal human plasma for ouabain-protein carrier complex. Ouabain-like biological activity was isolated in association with protein bands of 80, 50, and 25 kDa. These proteins appear to be human immunoglobulins or immunoglobulin-like because they are recognized by anti-human immunoglobulin antibodies, but not by anti-mouse immunoglobulin antibodies. The protein-containing fractions inhibit the binding of mAb 1-10 to immobilized ouabain, and with further purification on protein A, the immunoglobulin-like protein binds radioactive ouabain with an IC 50 of 200 to 600 nmol/L, but binds digoxin with 100-fold less affinity, suggesting specificity for ouabain or its isomer. Active protein fractions after purification on C 18 inhibit Na ϩ pump activity in human erythrocytes (IC 50Ϸ 4 nmol/L, ouabain equivalents), and this chromatography appears to dissociate the ouabain-like compound from the immunoglobulin protein(s). These immunoglobulin-like molecules may represent a subset of immunoglobulins (Յ0.5% of total protein A immunoglobulin) that function as a reservoir and delivery system for ouabain-like compounds in the modulation of human Na ϩ ,K ϩ -ATPase in vivo. (Hypertension. 2002;40:220-228.)

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Models for Prediction of Immunogenicity

Roggen¹

Immunogenicity of Biopharmaceuticals

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Sequences of antigenic epitopes of streptokinase identified via random peptide libraries displayed on phage 1 1Edited by J. A. Wells

Cited by 21 publications

References 46 publications

Leucine 42 in the Fibronectin Motif of Streptokinase Plays a Critical Role in Fibrin-independent Plasminogen Activation

Leucine 42 in the Fibronectin Motif of Streptokinase Plays a Critical Role in Fibrin-independent Plasminogen Activation

Ouabain-Binding Protein(s) From Human Plasma

Models for Prediction of Immunogenicity

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