Studies of the extracellular enzyme system of Aeromonas proteolytica

Abstract

An investigation into some of the properties of an endopeptidase of Aeromonas proteolytica was carried out. The endopeptidase was isolated by use of heat treatment at 60° for 20 minutes followed by gel filtration on Sephadex G-150 and ion exchange chromatography on DEAE A-50 or QAE A-50 Sephadex. Preparations were judged homogeneous by polyacrylamide gel electrophoresis ultracentrifugation, immunodiffusion, and immunoelectrophoresis before being subjected to other experiments. Sedimentation velocity and diffusion studies indicated an s°20 0f 3.44 S and a D°2o, w of 8.60 x 10�� cm² sec�¹. A partial specific volume of 0.711 cm³ g�¹ was calculated from amino acid analysis. These parameters were used to calculate a molecular weight of 34,600. Amino acid analysis indicated the enzyme to be composed of 316 amino acid residues. Atomic absorption spectrometry indicated the presence of 1 g-atom of zinc and a 2 g-atoms of calcium per mole of enzyme. A molar extinction of 55,681 at 280 nm was found. The pH optimum for activity against denatured hemoglobin was 8.0 and that for carbobenzoxyglycyl-L-phenylalanine was 7.5. Hydrolysis by carboxypeptidase A resulted in the release of five amino acids in stoichiometric quantities and is the basis for a proposed C-terminal sequence of -Thr-Ala-Phe -Tyr-Tyr. Aeromonas aminopeptidase hydrolysis was used to release amino acids from the N-terminus and is the basis to propose the N-terminal amino acid to be phenyalanine..