Über eine neue Carboxypeptidase (EC 3.4.2.?) aus der Milz

Abstract

An acid carboxypeptidase was demonstrated in bovine spleen. The specific activity in the cell-free supernatant of an homogenate was 6.0 mU/mg protein. The enzyme was purified 450-fold to a specific activity of 2.7 U/mg protein by ammonium sulphate precipitation, gel filtration and ion exchange chromatography. This purification removed the activities of cathepsins A, B, C and D, leucine aminopeptidase and catheptic carboxypeptidase. The substrate specificity of the acid carboxypeptidase from spleen was tested on more than 60 peptides and peptide derivatives, which were obtained by chemical synthesis. The enzyme hydrolysed tripeptides of the type Leu-Leu-X, in which the C-terminal amino acid was leucine, tyrosine, glutamic acid, valine, serine, alanine, aspartic acid or glycine, and tripeptides of the type X-Leu-Leu, in which the N-terminal amino acid was serine, glutamic acid, glycine, valine or histidine; the rate of hydrolysis decreased in the order of the listed amino acids. A series of non-leucine tripeptides was attacked very slowly. In tripeptides of the type Leu-Leu-X, in which the C-terminal amino acid is histidine, arginine or proline, the rate of cleavage falls to a few per cent compared with the standard substrate (Ser-Leu-Leu= 100%). Dipeptides were not attacked, tetrapeptides only to a very small extent There was no activity with substituted peptides or any of the unphysiological protease substrates. The acid carboxypeptidase has no proteolytic activity and is inactive towards proteins. Leucine and phenylalanine were, however, removed from their C-terminal position in proteolytic cleavage products of the oxidised B-chain of insulin. For the substrate Ser-Leu-Leu, the optimum pH in citrate buffer was 5.0, the optimum temperature for a 2 hr. incubation period was 50[degree] C, and the activation energy was 9800 cal/mol. The molecular weight of the enzyme was 5-8.104 (gel filtration). After separation of the subcellular particles of the spleen homogenate, the highest specific activity was found in the lysosome fraction. Michaelis constants for the tripeptides Ser-Leu-Leu, Leu-Leu-Leu, Leu-Leu-Val and Gly-Gly-Phe were 1.2, 1.6, 1.8 and 4.6.10"4m respectively. Metal ions, sulphydryl reagents and chelating agents in concentrations of 1.10-4M had no effect on enzyme activity. The acid carboxypeptidase described here is not identical with any of the carboxypeptidases reported so far. Since even substrates of catheptic carboxypeptidases are not attacked, the name "acid carboxypeptidase" is proposed.