Peptide mapping by polyacrylamide gel electrophoresis after cleavage at aspartyl-prolyl peptide bonds in sodium dodecyl sulfate-containing buffers

Rittenhouse, Judith; Marcus, Frank I.

doi:10.1016/0003-2697(84)90836-4

Cited by 86 publications

(31 citation statements)

References 17 publications

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“…For cleavage with HC1, the proteins were cleaved in mild acid (0.015 M) by heating at I10°C for 20 min. The reaction was stopped by rapid cooling in ice (Rittenhouse & Marcus, 1984). After incubating for different lengths of time, the gel slices were washed and equilibrated in 10~ glycerol, 2~ 2-mercaptoethanol, 3~ SDS in 625 mM-Tris-HCl pH 6.8 before loading on a second SDS~olyacryl-amide gel (15 ~).…”

Section: Methodsmentioning

confidence: 99%

Use of synthetic peptides to locate neutralizing antigenic domains on the fusion protein of respiratory syncytial virus

Bourgeois¹,

Corvaisier²,

Bour³

et al. 1991

Journal of General Virology

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Chemical and enzymic cleavages of the F~ subunit of the fusion (F) protein of respiratory syncytial (RS) virus showed that the sequence 184-Gly to 314-Trp reacted with neutralizing monoclonal antibodies (MAbs). Twelve synthetic peptides covering a part of this sequence were analysed for their immunoreactivity with neutralizing MAbs and anti-RS virus rabbit serum. Two sequential antigenic domains corresponding to amino acids 200 to 225 and 255 to 278 were defined with anti-RS virus rabbit serum. The peptides 205-225 and 259-278, belonging to these antigenic domains, inhibited binding to the F protein and the neutralizing activity of the anti-RS virus rabbit serum. One MAb (RS-348) reacted with peptides containing amino acids 200 to 225. Moreover, the peptide 205-225 induced an anti-peptide rabbit serum neutralizing RS virus in vitro. These results indicate that the sequence from residues 200 to 225 was present in one of the immunodominant sites of the F protein.

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Section: Methodsmentioning

confidence: 99%

Use of synthetic peptides to locate neutralizing antigenic domains on the fusion protein of respiratory syncytial virus

Bourgeois¹,

Corvaisier²,

Bour³

et al. 1991

Journal of General Virology

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“…However, it has been recognized for many years that Asp-Pro bonds are particularly susceptible to acid hydrolysis (28). In fact, one criticism of using the method of Laemmli when preparing samples for SDS-PAGE is that proteins are boiled in an acidic solution, a combination of conditions that can cause significant protein degradation (29). Although the Tris-based loading buffers commonly used in SDS-PAGE are near neutral pH (6.8), the solutions become significantly more acidic when heated.…”

Section: Acidic Hydrolysis Of Glu 298mentioning

confidence: 99%

Acidic Hydrolysis as a Mechanism for the Cleavage of the Glu298 → Asp Variant of Human Endothelial Nitric-oxide Synthase

Fairchild

Fulton

Fontana

et al. 2001

Journal of Biological Chemistry

153

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). We show that E298D-eNOS, as isolated from cells and in vitro, is susceptible to acidic hydrolysis, and the 100-kDa fragment can be generated ex vivo by increasing temperature at low pH. Importantly, cleavage of E298D was eliminated using a sample buffer system designed to limit acidic hydrolysis of AspPro bonds. These results argue against intracellular processing of E298D-eNOS and suggest that previously described fragmentation of E298D could be a product of sample preparation. We also found that eNOS turnover, NO production, and the susceptibility to cellular stress were not different in cells expressing WT versus E298D-eNOS. Finally, enzyme activities were identical for the respective recombinant enzymes. Thus, intracellular cleavage mechanisms are unlikely to account for associations between the exon 7 polymorphism and cardiovascular diseases.

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“…Since acetic acid cleavage of a labile Asp-Pro peptide bond (23,24) of proguanylin releases the bioactive COOHterminal guanylin-(80-94) (25), determination of the disulfide linkage is possible (10). The two topological isomers of guanylin-(80-94) give rise to a characteristic double peak in the HPLC pattern (5, 10) (Figure 3b).…”

Section: Purification Of Recombinant Proguanylinmentioning

confidence: 99%

Native and Recombinant Proguanylin Feature Identical Biophysical Properties and Are Monomeric in Solution

et al. 2002

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Guanylin, an intestinal peptide hormone and endogenous ligand of guanylyl cyclase C, is produced as the corresponding prohormone proguanylin. The mature hormone consists of 15 amino acid residues, representing the COOH-terminal part of the prohormone comprised of 94 amino acid residues. Here we report the recombinant expression and purification of proguanylin with its native disulfide connectivity, as well as the biophysical characterization of the recombinant and native protein. The comparison of recombinant and native proguanylin revealed identical biophysical and structural properties, as deduced from CZE, HPLC, and mass spectrometry, as well as NMR spectroscopy and CD spectroscopy at various temperatures and pH values. Exhaustive analytical ultracentrifugation studies were employed for protein concentrations up to the millimolar range to determine the association state of recombinant as well as native proguanylin, revealing both proteins to be monomeric at the applied solution conditions. As a result, a former identified close proximity between the termini of proguanylin is due to intramolecular interactions.The intestinal peptide hormone guanylin is an endogenous activator of guanylyl cyclase C (1). Activation of the cyclase markedly increases secretion of fluid and electrolytes into the intestinal lumen (2) by cGMP-mediated activation of the cystic fibrosis transmembrane conductance regulator (CFTR) (3, 4). For bioactivity, formation of the correct 1-3, 2-4 disulfide bonds of guanylin is necessary (1, 5). Even though guanylin possesses a unique cysteine connectivity, it is able to form two interconverting topological stereoisomers (6), with only one of them (A-form) showing significant biological effects (7).Guanylin is expressed as the corresponding prohormone containing 94 amino acid residues, with the mature hormone [guanylin-(80-94); numbering is according to the sequence of the prohormone] located at its COOH terminus, and is secreted into the intestinal mucosa and blood (8,9). It has been shown that an essential contribution of the prosequences of guanylin and the closely related uroguanylin is in the disulfide-coupled folding (10, 11). The prohormone, however, only shows negligible guanylyl cyclase C activating potency (10, 12). As an explanation for the inactivation of the bioactive COOH-terminal part of proguanylin, a shielding by its NH 2 terminus was suggested, since a close proximity of the NH 2 -and COOH-terminal regions of proguanylin was found (10). Since prouroguanylin and proguanylin show unusual elution behavior in size exclusion chromatography, they were assumed to be dimeric in solution (13). Nonetheless, no variation of the oligomerization state of native proguanylin was observed over the concentration range from 0.24 to 2.4 mM (10). As reversible association is concentration dependent, this result favors a monomeric state, but a very tight dimer cannot be excluded. An accurate determination of the oligomerization state, however, is a requirement for the correct interpretatio...

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Peptide mapping by polyacrylamide gel electrophoresis after cleavage at aspartyl-prolyl peptide bonds in sodium dodecyl sulfate-containing buffers

Cited by 86 publications

References 17 publications

Use of synthetic peptides to locate neutralizing antigenic domains on the fusion protein of respiratory syncytial virus

Use of synthetic peptides to locate neutralizing antigenic domains on the fusion protein of respiratory syncytial virus

Acidic Hydrolysis as a Mechanism for the Cleavage of the Glu298 → Asp Variant of Human Endothelial Nitric-oxide Synthase

Native and Recombinant Proguanylin Feature Identical Biophysical Properties and Are Monomeric in Solution

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