PLASMA MEMBRANE-ASSOCIATED AMINOPEPTIDASE ACTIVITIES IN CHLAMYDOMONAS-REINHARDTII AND THEIR BIOCHEMICAL-CHARACTERIZATION

High aminopeptidase (Apase) activities were found on intact unicellular algae cells. Several lines of evidence strongly indicate that the external Apases on Chlamydomonas reinhardtii (a green alga) cells, characterized in the present study, are plasma membrane-associated proteinases and not secreted in the cell wall or the surrounding medium. This is shown by enzyme activities also detected on a cell wall deficient mutant of C. reinhardtii and by the finding that in assay media and algal conditioned nutrient solutions, respectively, no Apase activities were found after removal of cells. In C. reinhardtii at least two in vivo Apases, one L-leucine-p-nitroanilide and one L-alanine-p-nitroanilide hydrolyzing enzyme (in vivo LeuNAase and AlaNAase, respectively) as well as one in vivo endoproteinase, capable of cleaving carboxybenzoylleucine-p-nitroanilide (CBZLeuNAase), were clearly distinguished by their pH optima for activity and characteristics towards various chemical compounds. In vivo LeuNAase, which cannot unequivocally classified as a metallo- or serine-type proteinase, showed optimum activities between pH 7 and 8.5, stimulation of activity by 1,10-phenanthroline (161%), 2-fold higher activity with L-phenylalanine-p-nitroanilide than with LeuNA and a K-m value of 40 mu M LeuNA. In vivo AlaNAase favored alkaline pH values, had a K-m value of 1.45 mM AlaNA and is probably a metallapeptidase as indicated by 2-fold enhancement of enzyme activity by 5 mu M Co2+ and strong inhibition with 1,10-phenanthroline. This enzyme was inhibited completely by a 30 min incubation with 10 mu M Hg2+ at room temperature, indicating sensitive SH-groups. In contrast, activity was stimulated 205% by 20 mM iodoacetate in the assay buffer. Both in vivo Apases were efficiently inhibited by 10 mM Pefabloc SC, a serine-type proteinase inhibitor and by two compounds, not yet described as proteinase inhibitors: methyljasmonate, a plant hormone, and dibucaine, a local anestheticum. The latter compound showed the most powerful inhibition an in vivo and in vitro LeuNAase of all reagents tested. From the distribution of Apase activities and characteristics in the cell, it is hypothesized that at least the LeuNAase dissociates easily from the plasma membrane during preparation of cell extracts and binds then unspecifically to various membrane fractions. In conclusion, this is the first report on the existence of external Apase activities on plant cells providing an easy-to-perform, rapid and reliable assay method for these enzymes.