The presence of the pyrrolidonyl peptidase activity in many tissues of pig, cow, rabbit, guinea-pig, rat, mouse, pigeon, hen and carp has been demonstrated. It was also found in some human tissues and in plants. The enzyme from the pigeon liver was partially purified and some of its properties were studied. By means of gel filtration of the pigeon and rabbit liver homogenates two enzyme fractions were separated and their molecular weight was estimated. The enzyme activity was inhibited by some ions, by -SH-blocking reagents, by pyrrolidone carboxylic acid and polyvinylpyrrolidone. Its specifity seemed t.0 be connected only with pyrrolidonecarboxylyl group, present in the substrate molecule.The presence of N-terminal pyrrolidonecarboxylyl group in some peptides and proteins has already been described by many mthors [l-4]. This group is synthesized enzymatically by cyclization of glutaminyl-tRNA to produce pyrrolidonecarboxylyltRNA and then transferred to the newly forming peptide chain [5].It is known that pyrrolidonecarboxylyl peptide bond is not hydrolysed by classical peptidyl hydrolases and therefore it is interesting to find a specific enzyme catalysing this reaction. Recently, the presence of this enzyme named pyrrolidonyl peptidase (and also known as pyrrolidonecarboxylyl peptidase [9]) has been described in Pseudomoms fluorescem [i], in Bacillus subtilis [6] and in many strains of Klebsiella, Citrobacter, Enterobacter, Arizona, Neisseria, Streptococcus and Stuphylococcus [7].I n this communication the presence of pyrrolidonyl peptidase in mammalian, bird, fish, plant and human tissues has been described. Some properties of the peptidase from rabbit and pigeon liver have been studied and also partial purification of it presented.A part of results contained in this communication was presented a t the 6th FEBS Meeting in Madrid, 1969 [S].
The synthesis of L-pyrrolidonyl-a-and /?-naphthylamides has been described. The /?-naphthylamide was used for the colorimetric determination of pyrrolidonyl peptidase activity. This enzyme was purified from Bacillus subti&? by precipitation with ammonium sulphate, using a DEAE-cellulose column and finally on a Sephadex G-200 column. The activity of the purified enzyme preparation was 530-fold that of the crude homogenate. No other peptidase activity in this preparation was found. It was demonstrated that the enzyme liberates pyrrolidonecarboxylic acid from human seromucoid. The results obtained suggest that pyrrolidonyl peptidase is an SH-enzyme. The occurrence of the enzyme in some groups of gram-positive and gram-negative bacteria was noted.Pyrrolidonecarboxylic acid can be formed enzymetically in organiams either by cyclization of free glutamic acid [1,2] or by cyclization of y-glutamyl [3,4] or glutaminyl [5] residues in peptides. It was considered of interest to search for a pyrrolidonecarboxylic acid liberating-enzyme (pyrrolidonylpeptidase) which attacks peptides and proteins with pyrrolidonyl residue in the N-terminal position. Having obtained such an enzyme, the amount of pyrrolidonecarboxylic acid and the significance of this acid bound to the peptide chain could be studied.To assay the pyrrolidonecarboxylic acid liberating-enzyme, we have synthesized new pyrrolidonylnaphthylamides as chromogenic substrates. It was observed that L-pyrrolidonyl-p-naphthylamide is readily hydrolyzed by an enzyme present in some bacteria. This enzyme has been purified from Bacillus subtilis and its properties and specificity have been described. This paper was presented at the 5th FEBS meeting in Prague, 1968 [5aJ
MATERIALS AXD METHODSChloroacetyl-p-naphthylamide and glycyl-pnaphthylamide were prepared according to Gomori Protein was determined by the colorimetric method of Lowry et al. [ll], and crystalline bovine serum albumin was used as a standard.For analytical purposes, paper high voltage electrophoresis was performed in acetic acid-pyridine buffer a t pH 3.6 or 6.5, and petroleum was used as cooling medium. Amino acids and peptides on the paper were localized by means of 0.3O/, ninhydrin solution in 800/, isopropanol containing 3'31, collidine. Pyrrolidonecarboxylic acid and peptides were localized by the chlor-starch-iodine method [12].
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