The interrelationship of aminopeptidase isoenzymes produced by Aeromonas proteolytica

Abstract

A previously unreported form of aminopeptidase has been isolated from culture filtrate of Aeromonas proteolytica by procedures in which autolytic and proteolytic degradation are deterred. The pH optimum, pH of stability, low temperature kinetics, susceptibility to inhibition and immunological reactivities are similar to those of Aeromonas aminopeptidase (designated herein as AAP-I) previously isolated in this laboratory. However, the new form (AAP-II) has a larger molecular weight than the previously isolated enzyme and isoelectric focusing indicates that that the isoelectric pH of the new enzyme is 0.3 PH unit greater than that of AAP-1. Analysis of AAP-II revealed lysine to be the NHâ‚‚-terminal amino acid whereas the NHâ‚‚-terminal of AAP-II is methionine. NHâ‚‚-terminal analysis of AAP-II that has been stored, implied the possibility of proteolytic degradation, and several intermediate protein bands observed after gel electrophoresis experiments suggest a cascade type process which terminates in the production of the very stable AAP-1. Thin layer chromatography of dansyl derivatives and amino acid analysis confirm that amino acids or peptides are released upon conversion of AAP-II to AAP-I. In the presence of low concentrations of inhibitors of the aminopeptidase, limited proteolysis occurred; however, in the presence of Aeromonas endopeptidase total conversion of AAP-II to AAP-I rapidly occurred. A two-dimensional gel electrophoresis system was developed to detect these conversions within the complex mixture of proteins in the culture filtrate and it should be useful in determining a number of changes in proteins that can be brought about by chemical or physical treatments which alter size, charge, or shape of the protein.