A method for purification of peptides from hydrolysates of proteins modified by chemically active analogues of substrates containing cis-diol groups

Abstract

A simple and rapid column procedure is described for the isolation from protein hydrolysates of peptides containing covalently bound substrate analogues with cis-diol groups. The method is based on complex formation between the cis-diol groups of peptide-bound compounds and dihydroxyborylic groups of a dihydroxyborylaminoethyl cellulose column. The method is useful for isolation of peptide(s) located in or near the active centre of enzymes after their affinity labelling by chemically active analogues of natural substrates like ribonucleotides, sugars, etc.

Phenylalanyl‐tRNA synthetase from escherichia coli MRE‐600

“…We observed activation in the presence of nucleowere found also in tryptophanyl-tRNA synthetase from beef pancreas [6].…”

“…All p-and yanilidates of nucleotides were obtained by condensation of nucleotides with amines in water in the presence of water-soluble carbodiimide. The synthesis and characterisation of these compounds were detailed in [6].…”

“…It was shown in [6] that some nucleotide mono-, di-and triphosphates as well as ~-anilidates of diphosphates and y-anilidates of triphosphates exhibit some activation properties at low concentration in the reaction catalyzed by tryptophanyl-tRNA synthetase from beef pancreas.…”

P1, P3‐bis(5′‐adenosyl)triphosphate (Ap3A) as a substrate and a product of mammalian tryptophanyl‐tRNA synthetase

“…The maximal reaction rate (Vmax) for Ap3A is about 113 of Vm,x for ATE This difference may be related to the difference of the reaction products (PPi in the case of ATP and ADP in the case of Ap3A): it is known that ADP is a TrpRS inhibitor non-competitive with ATP [16].…”

Photoaffinity Leabeling of Chloroplastic and Cytoplasimic Leycly‐rRNA Synthetases of Euglena gracilis, by the γ‐(p‐Azidoanilidae) of ATP

“…Addition of ATP and leucine during ultraviolet-inactivation by AzAn ATP prcvents covalent binding of the analog to LRS 1. Moreover, the inhibitor binds only to two out of 49 total tryptic peptides which were isolated according to [27]. These results have been published in a preliminary form [28] (full paper in preparation).…”