Distribution and Characteristics of Aminoacyl β-Naphthylamidase Activities in Wheat Seedlings

Abstract

Gel electrophoretic studies have revealed that crude extracts from various tissues of wheat seedlings contain two major enzymes capable of hydrolysing aminoacyl �-naphthylamides. A third enzyme which exclusively hydrolyses the �-naphthylamides of the imino acids proline and hydroxyproline has also been demonstrated in wheat leaves. The pH optimum for �-naphthylamidase activity against phenylalanine �-naphthylamide in crude extracts was 7.4. The two major enzymes differ with respect to their substrate specificities; the more anionic enzyme, APl, hydrolyses a relatively narrow range of hydrophobic aminoacyl substrates including the �-naphthyIamides of leucine, phenylalanine, methionine, tyrosine and tryptophan, while the enzyme of lower electrophoretic mobility, AP2, hydrolyses a broad range of substrates. The two enzymes also differ in their sensitivity to the metal chelator 1,10-phenanthroline. The AP2 enzyme from wheat, like that from pea, appears to be a metallo-enzyme, but APl does not show any sensitivity to phenanthroline. The results of developmental studies performed with wheat seedling tissues are consistent with the view that the naphthylamidase enzymes function as aminopeptidases in vivo. A close association was observed between total enzyme activity and soluble protein content, with the highest naphthylamidase activities being found in actively growing tissues.

Characterization and Subcellular Localization of Aminopeptidases in Senescing Barley Leaves

“…This same trend was reported by Waters and Dalling (29), showing an increased ,3-naphthylamidase activity in wheat seedling leaves to day 7 followed by a continuous decrease. In oat leaves placed in the dark, AP activity against three substrates declined steadily (12).…”

“…Significant inhibition occurred in the presence ofboth strong chelating (1,10-phenanthroline and Bath) and sulfhydryl (pCMB) type inhibitors. Other investigators have reported similar results; three maize APs (27) and other APs in pea (4,9), wheat (29), and Euonymus (24) also showed significant inhibition by both sulflhydryl inhibitors and chelating agents.…”

“…Waters et al. (29,30) reported that AP activity, using Ala-, Phe-, and Pro-,B-NA, was nearly equally distributed between chloroplasts and cytoplasm of wheat leaves. They also found no AP activity associated with vacuoles.…”

Intracellular Localization of Peptide Hydrolases in Wheat (Triticum aestivum L.) Leaves

“…Although considerable quantities of mitochondria and peroxisomes were occluded within the intact chloroplast band, chloroplast-free zones of peroxisomes (fractions 5-8) and mitochondria (fractions 13-16) were also located in the gradient. The cytoplasm, identified by glucose-6-P dehydrogenase (fractions [26][27][28][29], was contained in the supernatant on top of the sucrose gradient. The supernatant also contains the contents of broken organelles, including the vacuoles, which are too large to pass intact through the 20-,um mesh used to break the protoplasts.…”

“…Protein degradation has been envisaged as a process initiated by endopeptidase action and continued through the action of exopeptidases, including aminopeptidases, carboxypeptidase, and di-and tri-peptidases to eventually release amino acids for storage and/or transport (24). Characterization of these enzymes (5,15,22,29) has shown that, despite their common role in protein breakdown, they have few similarities with respect to the conditions required for optimal in vitro activity. These differences, particularly differences in optimum assay pH, have led to suggestions of enzyme compartmentalization as a means of regulating protein degradation, since the plant cell contains a number of localized pH zones by virtue of its various organelles.…”