MOLECULAR AND IMMUNOLOGICAL CHARACTERIZATION OF LEUCINE AMINOPEPTIDASE IN ARABIDOPSIS-THALIANA - A NEW ANTIBODY SUGGESTS A SEMI-CONSTITUTIVE REGULATION OF A PHYLOGENETICALLY OLD ENZYME

Plants of Arabidopsis thaliana probably contain only a single leucine aminopeptidase (LAP) gene and are therefore good candidates for investigating the still unknown function of LAPs in plants. The cDNA clone PM25 [1], encoding a A. thaliana leucine aminopeptidase of 520 amino acids with a calculated molecular mass of 54 506 Da, was expressed in Escherichia coli with a N-terminal sequence extension as a protein of molecular mass 57 kDa. This polypeptide was purified and used to raise an anti-LAP antiserum. The antiserum recognized a single band of 54 kDa on nitrocellulose-blots following denaturating gel electrophoresis of A. thaliana protein. Zymogram analysis of the native enzyme using L-leucine P-naphthylamide as substrate indicated a hexameric structure of 320 kDa for the active complex in plants. Immunoblot analysis of LAPs in several di- and monocotyledonous species indicated a subgroup of a phylogenetically old enzyme. LAP protein in A, thaliana can be detected at different developmental stages, in different organs and after treatment of plants with phytohormones or by wounding. Except for slight variations in the accumulation of LAP during development and organ specificity, the data indicate that LAP is present during all stages of development in A. thaliana.