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 invest igating the still unknown function of LAPs in plants. The cDNA clone P M25 [1], encoding a A. thaliana leucine aminopeptidase of 520 amino ac ids with a calculated molecular mass of 54 506 Da, was expressed in Es cherichia coli with a N-terminal sequence extension as a protein of mo lecular mass 57 kDa. This polypeptide was purified and used to raise a n 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-l eucine 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 detect ed at different developmental stages, in different organs and after tr eatment of plants with phytohormones or by wounding. Except for slight variations in the accumulation of LAP during development and organ sp ecificity, the data indicate that LAP is present during all stages of development in A. thaliana.