Purification and Characterization of a Novel Aminopeptidase, Plastidial Alanine-Aminopeptidase, from the Cotyledons of Etiolated Sugar Beet Seedlings

Abstract

During prolonged dark growth of sugar beet (Befa vulgaris L.) seedlings, etioplasts, rapidly after the proplastid-etioplast transition, undergo a degenerative process characterized by ultrastructural modifications, protein loss, and the decrease of carotenoid and chlorophyll accumulation upon illumination. Two plastidial aminopeptidase activities were identified as early markers of this degenerative process (A. El Amrani, 1. Couée, 1.-P. Carde, 1.-P. Caudillère, P. Raymond [19941 Plant Physiology 106: 1555-1565). l h e present study focuses on one of these markers and describes the purification to homogeneity and characterization of plastidial alanine-aminopeptidase. This nove1 aminopeptidase was found to be a metallotype naphthylamidase particularly active with alanyl, arginyl, and leucyl substrates. Its plastidial location was confirmed by immunofluorescence with polyclonal antibodies against the purified enzyme. Its physico-chemical and enzymic properties are discussed with respect to other higher plant aminopeptidases and to its potential functions during prolonged dark growth.

Targeting and localization of wound-inducible leucine aminopeptidase A in tomato leaves

“…These data indicate that LAP-A and LAP-N join the group of plastid-localized aminopeptidases that have been previously characterized biochemically (Waters et al 1982;Liu and Jagendorf 1986;Thayer et al 1988;Amrani et al 1995;Desimone et al 2000) or predicted from genome sequence initiatives (Richly and Leister 2004;Millar et al 2006;Walling 2006). The immunolocalization of LAP-A proteins in the plastids is in contrast with previous cell fractionation studies, where LAP-A proteins were primarily detected in the soluble cytosolic fraction, and only a small quantity was found within the chloroplasts (Gu et al 1996a).…”

Protein stability and degradation in chloroplasts

“…Peptidases capable of cleaving only short peptides were also isolated from plastids. An alanyl peptidase was isolated from etioplasts [31 ] and an arginyl peptidase was purified from chloroplasts [26]. Both of them have similar subunit molecular mass, 75-80 kDa, and they both occur as dimers, but their expression pattern is different and their physiological significance is not clear.…”

Involvement of exopeptidases in dehydration tolerance of spring wheat seedlings

“…The Ala-β-NA specific aminopeptidase seems to be metalloenzyme that is inhibited by metal chelators (1,10-phenantroline and EDTA). The efficient inhibitory effect obtained with pCMB with concomitant lack of inhibitory effect of E-64 on alanine aminopeptidase implicated the involvement of Cys residues in retain the appropriate enzyme conformation (El-Amrani et al 1995, Ogiwara et al 2005. The second wheat seedling aminopeptidase seems to be cysteine enzyme, as measured by the inhibitory effect of pCMB and E-64.…”