Aminosäure-<i>p</i>-nitroanilide als Substrate für Aminopeptidasen und andere proteolytische Fermente

Tuppy, H.; Wiesbauer, Ulrike; Wintersberger, Erhard

doi:10.1515/bchm2.1962.329.1.278

Cited by 224 publications

(46 citation statements)

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“…The synthesis of p-nitroanilides of S-methylcysteine, Sethylcysteine and methionine will be published elsewhere. The analytical data of protected and free amino acid p-nitroanilides which have not been published so far are shown in Table 1 [21,22].…”

Section: Substratesmentioning

confidence: 99%

p‐Nitroanilides of 3‐Carboxypropionyl‐peptides

Kasafírek

Fric²,

Slabý³

et al. 1976

European Journal of Biochemistry

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Fourteen 3-carboxypropionyl-tripeptide-p-nitroanilides of the general formula 3-carboxypropionyl-alanyl-alanyl-Y-p-nitroanilide (Y = glycine, norvaline, S-methylcysteine, valine, norleucine, S-ethylcysteine, methionine, leucine, isoleucine, phenylalanine, tyrosine, S-benzylcysteine, C"-phenylglycine, and proline) were synthesized and their cleavage by elastase, trypsin, and chymotrypsin (Km, kcat and k,at/Km) was determined.The significance of amino acid residues in the position of Y was evaluated firstly with respect to their structure (topographically), and secondly with respect to their free energy (thermodynamically). The alanine residue substrate was cleaved best by elastase, the phenylalanine substrate by chymotrypsin. Trypsin cleaved two substrates only, that is those containing a phenylalanine and a tyrosine residue.The optimum length of the elastolytic substrates was studied in a series of N-3-carboxypropionyl-(Ala),-p-nitroanilides (n = 1, 2, 3, 4, 5), N-3-carboxypropionyl-(Gly),-p-nitroanilides (n = 1, 2, 3), and in p-nitroanilides of fatty acids with two to seven carbon atoms. Elastase cleaved tri, tetra, and pentapeptides of alanine.p-Nitroanilides of the glycine series, as well as p-nitroanilides of fatty acids were not cleaved. 3-Carboxypropionyl-tetra-alanine-p-nitroanilide was the most suitable substrate so far found for elastase cleavage; it is not cleaved by trypsin nor chymotrypsin. The optimum distance between Y and the terminal anionic carboxyl residue was found to be 1.8 nm in elastolytic substrates.Pancreatic elastase has lately been the subject of interest concerning the mechanism of its effect, and also concerning its presumed participation in the pathogenesis of some diseases, such as pancreatitis [l -31, lung emphysema [4,5], and atherosclerosis [6].A series of various peptides has already been prepared to elucidate and determine the optimum amino acid sequences in synthetic substrates [7 -101. The bond between C-terminal alanine and the corresponding amide or ester bond is the most easily split. Succinyl-alanyl-alanyl-alanine-p-nitroanilide, described in the preceding communication, has been shown to be the most specific substrate [ll]. (The positions of amine acid residues, discussed below, are defined in this peptide as P4-P3-PI-P1-Pi .) The principal reason for the synthesis of the anionic substrates has been perceived as the proof of electrostatic interactions between the substrate and elastase (in the sense of Gertler's conception of the natural substrate bonding) [12]. Electrostatic induction brings about a change in the elastase conformation according to Koshland's theory of 'induced fit' [13]. Since this change is caused also by natural ionogenic substances (salts of cholic acids) [14] and thus probably occurs under physiological conditions, it has been considered adequate to modify our type of ionogenic peptide and to prove 'optimalization' of the amino acid residue in the position PI [15]. The use of p-nitroaniline as chromogenic residue permits the cleavage between the pos...

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Section: Substratesmentioning

confidence: 99%

p‐Nitroanilides of 3‐Carboxypropionyl‐peptides

Kasafírek

Fric²,

Slabý³

et al. 1976

European Journal of Biochemistry

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“…[14]. Leucine-isoleucinevaline binding protein (LIV-protein) from E. co/i was isolated as in 115,161 by SD.…”

Section: Methodsmentioning

confidence: 99%

Role of intestinal brush border membrane aminopeptidase N in dipeptide transport

et al. 1984

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The kinetics of uptake of radioactive label from [U‐14C]Gly, L‐[4,5‐3H]Leu and the dipeptide [14C]Gly—L‐[4,5‐3H]Leu by the brush border membrane vesicles of porcine small intestine have been studied. The effect of aminopeptidase N inhibitors and leucine‐binding protein on accumulation rates has also been tested. Comparison of the kinetic parameters for uptake and hydrolysis of Gly—L‐Leu makes it possible to conclude that the dipeptide transfer includes two conjugated steps, viz., hydrolysis catalysed by aminopeptidase N and transport of the resultant free amino acids by a specific carrier.

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“…The enzyme was treated with Dip-F (100 pg/ml for 30 min) to inactivate traces of endopeptidases which might be present in the preparation. Clostridial aminopeptidase was prepared as described by Kessler and Yaron [8]. All the reagents used were of the highest purity commercially available.…”

Section: Methodsmentioning

confidence: 99%

“…interruption of the long-range interaction between the anthraniloyl fluorophore and the quenching nitrobenzyl group. The same compounds were shown to act as competitive inhibitors of the extracellular aminopeptidase from Clostridium histolyticum, which is very similar to leucine aminopeptidase in its substrate specificity [8].…”

mentioning

confidence: 98%

Intramolecularly‐Quenched Fluorescent Peptides as Fluorogenic Substrates of Leucine Aminopeptidase and Inhibitors of Clostridial Aminopeptidase

Carmel

Kessler

Yaron

1977

European Journal of Biochemistry

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Fluorogenic oligopeptide derivatives of the type Lys(ABz)-X-ONBzl, where ABz is o-aminobenzoyl (anthraniloyl), X stands for Ala, Phe, or Ala-Ala, and ONBzl is p-nitrobenzyloxy, were synthesized and shown to be hydrolyzed by leucine aminopeptidase. The hydrolysis is accompanied by an increase in fluorescence due to disruption of the intramolecular quenching of the fluorescent anthraniloyl moiety by the nitrobenzyl ester group. The spectral characteristics of the compounds are not consistent with an energy transfer mechanism according to Forster, therefore the quenching is assumed to be caused by a direct encounter between the quenching and the fluorescent groups. The change in fluorescence that accompanies the enzymic hydrolysis of the first peptide bond was used for quantitative measurement of the activity of leucine aminopeptidase and for the determination of some of its kinetic parameters. A bacterial aminopeptidase from Clostridium histolyticum that is very similar to leucine aminopeptidase in its substrate specificity did not hydrolyze the above peptide derivatives. The hydrolysis of leucine p-nitroanilide by this enzyme was found to be inhibited by the three peptides and the corresponding inhibition constants were determined.Spectrophotometric methods commonly used for assaying the activity of aminopeptidases and for studying their substrate specificity are based on the hydrolysis of p-nitroanilides [l] or P-naphthylamides [2] of amino acids, leucine derivatives being the most widely used. In these substrates the chromophores are directly engaged in the enzymically affected bond, the cleavage of which results in significant spectral changes, due to the conversion of the aromatic amide into an aromatic amine. Since the interaction of aminopeptidases with natural substrates involves the recognition of more than one amino acid residue [3,4], it should be of advantage to develop derivatives of oligopeptides rather than of amino acids as chromogenic substrates. For this purpose, however, a different approach is required that would allow spectrophotometric monitoring of the hydrolysis of an N-terminal peptide bond in spite of the requirement that the chromophore probe cannot be directly engaged in the Ahhrrviations. Abbreviations for amino acid derivatives and peptides follow the recommendations of IUPAC-IUB Commission on Biochemical Nomenclature, see Eur.

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Aminosäure-p-nitroanilide als Substrate für Aminopeptidasen und andere proteolytische Fermente

Cited by 224 publications

References 0 publications

p‐Nitroanilides of 3‐Carboxypropionyl‐peptides

p‐Nitroanilides of 3‐Carboxypropionyl‐peptides

Role of intestinal brush border membrane aminopeptidase N in dipeptide transport

Intramolecularly‐Quenched Fluorescent Peptides as Fluorogenic Substrates of Leucine Aminopeptidase and Inhibitors of Clostridial Aminopeptidase

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