N-anthraniloylation converts peptide p-nitroanilides into fluorogenic substrates of proteases without loss of their chromogenic properties

Bratovanova, Emilia K.; Petkov, Dimiter D.

doi:10.1016/0003-2697(87)90029-7

“…The results obtained with the substrate acyl-Ala-Ala-Val-Tyr-His-Gln-Ala-Ala-NH, indicated that this peptide could form the basis for designing intramolecularly quenched substrates (Yaron et al, 1979). Hydrolysis of this type of substrate can be followed by continuously monitoring the changes in fluorescence and affords a more rapid and sensitive assay for proteinases (Bratovanova and Petkov, 1987). In the present case, O-aminobenzoyl-Ala-Ala-Val-Tyr-His-Gln-Ala-Ala-NH-Np has been chosen as a potential fluorogenic substrate.…”

Section: Resultsmentioning

confidence: 99%

Purification of Turnip Mosaic Potyvirus Viral Protein Genome-Linked Proteinase Expressed in Escherichia coli and Development of a Quantitative Assay for Proteolytic Activity

Ménard¹,

Chatel²,

Dupras³

et al. 1995

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The 49-kDa, nuclear inclusion a-like, viral protein genome-linked proteinase (VPg-Pro) of turnip mosaic potyvirus (TuMV) was expressed in Escherichia coli. The protein was produced in a soluble form at high levels and was active, as demonstrated by intermolecular cleavage of the polymerase capsid protein (Pol-CP) substrate. The VPg-Pro was purified by metal-chelation and ion-exchange chromatographies. Two forms of VPg-Pro, which differed in molecular masses, were obtained during isolation; their identities were confirmed by immunoblot analysis and N-terminal amino acid sequencing. Data indicated that cleavage took place at a site near the C-terminus of VPg-Pro and was the result of the proteolytic activity of the viral protein. The purified proteinase retained enzymic activity on its natural substrate (Pol-CP) and was also capable of hydrolysing the synthetic peptide acyl-Ala-Ala-Val-Tyr-HisGln-Ala-Ala-NH,, derived from the consensus cleavage site for the TuMV polyprotein. Analysis by mass spectrometry of the two fragments resulting from this reaction indicated that cleavage took place between the Gln and Ala residues, as expected. A fluorogenic derivative of this peptide was hydrolysed by VPgPro, affording a convenient quantitative assay for intermolecular proteolytic activity, and was used to determine the pH-activity profile. The availability of large quantities of pure proteinase and of a rapid and sensitive assay will permit detailed kinetic and structural studies which are essential to obtain a better understanding of the mode of action of this and related viral proteinases, such as the 3C proteinase of picornaviruses.

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“…The complex specificity of proteases is clear from the diversity of the substrates and methods that have been developed for their assays: spectrophotometry (Feder, 1968), fluorescence (Auld and Prescott, 1983;Bratovanova and Petkov, 1987;Kuromizu et al, 1985;Tisljar and Denker, 1986), HPLC (Green, 1986;Kuwada and Katayama, 1984;Nagatsu et al, 1985), ELISA (Ohlsson et al, 1986;Yoshioka et al, 1987), and methods using gel-entrapped substrates (Brown et al, 1982;Kelleher and Juliano, 1984;Nagase and Woessner, 1980) or luminogenic and chromogenic substrates (Branchini et al, 1980). When there is no information about the proteases involved, the choice of the method and substrate becomes an even larger problem.…”

Section: Introductionmentioning

confidence: 99%

Proteolytic activity in infected and noninfected insect cells: Degradation of HIV-1 Pr55gag particles

Cruz

¹

,

Martins

²

,

Alves

³

et al. 1999

Biotechnol. Bioeng.

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“…The results obtained with the substrate acylAla-Ala-Val-Tyr-His-Gln-Ala-Ala-NH, indicated that this peptide could form the basis for designing intramolecularly quenched substrates (Yaron et al, 1979). Hydrolysis of this type of substrate can be followed by continuously monitoring the changes in fluorescence and affords a more rapid and sensitive assay for proteinases (Bratovanova and Petkov, 1987). In the present case, O-aminobenzoyl-Ala-Ala-Val-Tyr-His-Gln-AlaAla-NH-Np has been chosen as a potential fluorogenic substrate.…”

Section: Resultsmentioning

confidence: 99%

Purification of Turnip Mosaic Potyvirus Viral Protein Genome‐Linked Proteinase Expressed in Escherichia coli and Development of a Quantitative Assay for Proteolytic Activity

Ménard

¹

,

Chatel

²

,

Dupras

³

et al. 1995

European Journal of Biochemistry

View full text Add to dashboard Cite

The 49-kDa, nuclear inclusion a-like, viral protein genome-linked proteinase (VPg-Pro) of turnip mosaic potyvirus (TuMV) was expressed in Escherichia coli. The protein was produced in a soluble form at high levels and was active, as demonstrated by intermolecular cleavage of the polymerase capsid protein (Pol-CP) substrate. The VPg-Pro was purified by metal-chelation and ion-exchange chromatographies. Two forms of VPg-Pro, which differed in molecular masses, were obtained during isolation; their identities were confirmed by immunoblot analysis and N-terminal amino acid sequencing. Data indicated that cleavage took place at a site near the C-terminus of VPg-Pro and was the result of the proteolytic activity of the viral protein. The purified proteinase retained enzymic activity on its natural substrate (Pol-CP) and was also capable of hydrolysing the synthetic peptide acyl-Ala-Ala-Val-Tyr-HisGln-Ala-Ala-NH,, derived from the consensus cleavage site for the TuMV polyprotein. Analysis by mass spectrometry of the two fragments resulting from this reaction indicated that cleavage took place between the Gln and Ala residues, as expected. A fluorogenic derivative of this peptide was hydrolysed by VPgPro, affording a convenient quantitative assay for intermolecular proteolytic activity, and was used to determine the pH-activity profile. The availability of large quantities of pure proteinase and of a rapid and sensitive assay will permit detailed kinetic and structural studies which are essential to obtain a better understanding of the mode of action of this and related viral proteinases, such as the 3C proteinase of picornaviruses.

show abstract

N-anthraniloylation converts peptide p-nitroanilides into fluorogenic substrates of proteases without loss of their chromogenic properties

Cited by 20 publications

References 15 publications

Purification of Turnip Mosaic Potyvirus Viral Protein Genome-Linked Proteinase Expressed in Escherichia coli and Development of a Quantitative Assay for Proteolytic Activity

Purification of Turnip Mosaic Potyvirus Viral Protein Genome-Linked Proteinase Expressed in Escherichia coli and Development of a Quantitative Assay for Proteolytic Activity

Proteolytic activity in infected and noninfected insect cells: Degradation of HIV-1 Pr55gag particles

Purification of Turnip Mosaic Potyvirus Viral Protein Genome‐Linked Proteinase Expressed in Escherichia coli and Development of a Quantitative Assay for Proteolytic Activity

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