| 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 10⁻⁷ 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.. |
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