Kinetic Characterization of Affinity Chromatography Purified Clostripain

Ullmann, Dirk; Jakubke, Hans‐Dieter

doi:10.1515/bchm3.1994.375.2.89

Cited by 6 publications

(7 citation statements)

References 10 publications

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“…The kinetic data of these calculations are summarized in Table 1. The kinetic parameters for Mal-Tyr-Arg-OEt are similar to those obtained in previous experiments (Ullmann and Jakubke, 1994a). In accordance with previously published data (Ogle and Tytell, 1953;Cole ef a/., 1971) the results in Table 1 show a two to three orders of magnitude greater specificity of clostripain for ester substrates with arginine residues at P!…”

Section: Subsite Specificity Studies On the Unusual Cysteine Proteasesupporting

confidence: 91%

“…The hydrolysis of susceptible substrates is primarily limited to the carboxyl side of arginine residues (Ogle and Tytell, 1953;Labouesse and Gros, 1960). In order to investigate the primary subsite specificity of clostripain, several substrates and inhibitors were applied in kinetic characterization and inhibition studies (Cole et a/., 1971;Porter et a/., 1971;Rainey, 1972;Gilles ei a/., 1979;Ullmann and Jakubke, 1994a). Because of the acceptance of proline in the Pj position (nomenclature according Schechter and Berger, 1967) clostripain has been interesting for protease-catalyzed peptide synthesis since most proteolytic enzymes do not accept proline at this position.…”

Section: Subsite Specificity Studies On the Unusual Cysteine Proteasementioning

confidence: 99%

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Short Communication

1997

Biological Chemistry

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The luciferin-binding protein (LBP) from the dinoflagellate, Gonyaulax polyedra, is regulated by a circadian clock at the translational level. A 22-nucleotide long interval in the Ibp 3' untranslated region, which contains seven UG-repeats, was characterized as a circadian c/s-acting element, to which a clock controlled factor (CCTR) binds. Recently we have found that the phylogenetically distant green alga, Chlamydomonas reinhardtii, contains a CCTR analog, called Chlamy 1. Here we show that the flanking nucleotides surrounding the UG-repeats are required for high binding activity of CCTR and Chlamy 1. The absence of three or more UG-repeats abolishes binding with both proteins.

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Section: Subsite Specificity Studies On the Unusual Cysteine Proteasesupporting

confidence: 91%

Section: Subsite Specificity Studies On the Unusual Cysteine Proteasementioning

confidence: 99%

Short Communication

1997

Biological Chemistry

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“…Clostripain was obtained from H chst AG (Frankfurt/ Main, Germany) and purified according to the procedure given by Ullmann and Jakubke (1994). CE-running buffer (20 ΠΊΜ sodium citrate, pH 2.5) was purchased from Applied Biosystems (Weiterstadt, Germany) and TFE from Sigma (Deisenhofen, Germany).…”

Section: Methodsmentioning

confidence: 99%

Influence of Helix Formation oncis/transIsomerism of Xaa-Pro Bonds Flanking the Helical Segment

Meyer¹,

Drewello²,

Fischer³

1996

Biological Chemistry Hoppe-Seyler

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The trifluoroethanol (TFE)-induced formation of ahelical structures in the peptide hormone calcitonin (salmon) was studied using limited proteolysis combined with capillary zone electrophoresis. A low TFE content in TFE/buffer mixtures was insufficient to introduce secondary structure, but two Lys-Leu bonds were found to have become inaccessible to proteolysis with clostripain. The influence of increasing helical degree of the Thr 6 -Lys 18 (or ThrMyr 22 in the trans conformation) segment on the cis/trans isomerization of the adjacent Tyr^-Pro 23 peptide bond was examined by means of isomer specific proteolysis. Results indicate that the helix dipole does not influence the cis/-trans equilibrium distribution of the flanking Tyr 22 -Pro 23 bond but considerably increases its isomerization rate /r c^t ·

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“…Clostripain was obtained from Hoechst and purified as described previously (Ullmann and Jakubke, 1994a). The enzyme was activated for 4-5 h in the presence of 1.0 mM CaCl,, containing 2.5 mM dithiothreitol at a specific activity of 400 nkat/ml.…”

Section: Methodsmentioning

confidence: 99%

“…Stock solutions of the nucleophiles (500 mM and 50 mM) were prepared in 0.2 M sodium carbonate/sodium bicarbonate (pH 9.0) containing appropriate amounts of NaOH to neutralize hydrochlorides or trifluoroacetates and to deprotonate the carboxyl function in the case of nucleophiles with low solubility. The final acyl donor concentration was 2 mM and the nucleophile concentrations were 50, 10, 5, 2 and 1 mM, which were calculated as free nucleophile concentrations The preactivated enzyme stock solution was prepared daily and enzyme activity was checked spectrophotometrically on a Shimadzu UV-160A spectrophotometer (Ullmann and Jakubke, 1994a).…”

Section: Acyl-transfer Experimentsmentioning

confidence: 99%

The specificity of clostripain from Clostridium histolyticum

Ullmann¹,

Jakubke²

1994

European Journal of Biochemistry

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The S' subsite specificity of clostripain from Clostridium histolyticum was investigated by acyl transfer to libraries of amino acid amides, Ala-Xaa dipeptides, proline derivatives and pentapeptides using Nu-benzoyl-L-arginine ethyl ester as acyl donor. A pentapeptide library consisting of 29 pentapeptides with general structure Xaa-Ala-Ala-Ala-Gly, Ala-Xaa-Ala-Ala-Gly and Ala-Ala-XaaAla-Gly, where Xaa represents Gly, Ala, Pro, Leu, Phe, Asp, Glu, Arg and Lys, was prepared by solid-phase synthesis. The data analysis was performed by HPLC and evaluated by statistical algorithms. The nucleophile efficiency covers a range of more than three orders of magnitude. In the P; position, low specificity for amino acid amides and Xaa-(Ala),-Gly peptides was found, however, in the Pi position, positively charged amino acid residues are strongly preferred. The negatively charged side chains of aspartic acid and glutamic acid in the P: and Pi positions, respectively, show only poor nucleophilic behaviour. In the case of these amino acids, the S'-P' interactions depend significantly on their position of these residues in the peptide chain of the nucleophile. The transfer of aspartic acid and glutamic acid from P; -Pi increases the nucleophile efficiency by approximately two orders of magnitude. The aromatic side chains are not well accepted, especially in the case of PiPhe. Surprisingly, P:Gly leads to effective P'-S' interactions. However, the opposite result was obtained for PiGly. The high efficiency for Gly-NH, does not fit with the hydrophobicity structure/ activity relationships. In most cases, peptide chain elongation does not improve the nucleophile efficiency. The effective interaction of D-Leu-NH, with the S' subsite of clostripain emphasizes the fact that the nucleophile stereospecificity is not restricted to L-amino acids. The results with proline derivatives indicate remarkably different specificities of the S' binding site which can only be explained by conformational restraints. A positive cooperativity between P:Pro and PiGly and a negative cooperativity between Pi Pro and PiPhe was observed. The arrangement of three proline residues next to each other represents a favourable conformation for effective enzyme-nucleophile interactions.The cysteine protease clostripain (Clostridium histolyticum proteinase B) from the culture filtrate of C. histolyticum is well known for its restricted substrate specificity, the cleavage of Arg-Xaa bonds, including proline in the P; position (subsite nomenclature of Schechter and Berger, 1967) (Ogle andTytell, 1953 ;Labouesse and Gros, 1960; Mitchell, 1964). For this reason this enzyme is important both in sequence analysis and in enzymic peptide synthesis, because proline is normally not well accepted in the P: position by the majority of proteases. Furthermore, a precise characterization of clostripain specificity is important if selective inhibitors are to be designed.Although S subsites have been studied in detail (Cole et al., 1971;Porter et al., 1971;Gilles et al., 1979;

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Kinetic Characterization of Affinity Chromatography Purified Clostripain

Cited by 6 publications

References 10 publications

Short Communication

Short Communication

Influence of Helix Formation oncis/transIsomerism of Xaa-Pro Bonds Flanking the Helical Segment

The specificity of clostripain from Clostridium histolyticum

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