Identification and biochemical characterization of plant acylamino acid-releasing enzyme.

Plant acylamino acid-releasing enzyme (AARE) catalyzing the N-terminal hydrolysis of N(alpha)-acylpeptides to release N(alpha)-acylated amino acids, was biochemically characterized using recombinant and native AAREs. A cDNA encoding a deduced Arabidopsis thaliana AARE (AtAARE) was cloned and sequenced. The deduced amino acid sequence encoded a 764 amino acid protein of 83.9 kDa, which was 31.8% identical with that of rat AARE. In particular, the proposed catalytic residues (Ser, Asp, and His) of AARE, called the "catalytic triad residues, " were completely conserved. Recombinant AtAARE was expressed in Escherichia coli and confirmed to be a functional AARE. Native AAREs were prepared from A. thaliana and cucumber (Cucumis sativus, L.) plants. Both native AAREs were tetrameric proteins of 350 kDa comprising four subunits of 82 kDa, and showed typical enzymological properties of other AAREs, i.e. sensitivity to diisopropyl fluorophosphate, an optimum pH of around 7.0, and an optimum temperature of 37 degrees C. Both the native and recombinant AAREs were immunochemically homologous. Intracelluar fractionation analysis showed that the AARE was mainly present in the stroma of chloroplasts. Native AARE degraded the glycated ribulose-1,5-bisphoshate carboxylase/oxygenase protein but not the native protein. Thus, plant AARE might be involved in not only catalysis of the N-terminal hydrolysis of N(alpha)-acylpeptides but also the elimination of glycated proteins.

Glycation by ascorbic acid causes loss of activity of ribulose-1,5-bisphosphate carboxylase/oxygenase and its increased susceptibility to proteases.

Glycation is a process whereby sugar molecules form a covalent adduct with protein amino groups. In this study, we used ascorbic acid (AsA) as a glycating agent and purified cucumber (Cucumis sativus L.) ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) as a model protein in chloroplast tissues, and examined effects of glycation on the activity and susceptibility of Rubisco to proteases. Glycation proceeded via two phases during incubation with AsA and Rubisco in vitro at physiological conditions (10 mM AsA, pH 7.5, 25 degrees C in the presence of atmospheric oxygen). At the early stage of glycation (phase 1), the amount of AsA attaching to Rubisco increased at an almost linear rate (0.5-0.7 mol AsA incorporated (mol Rubisco)(-1) d(-1)). By Western blotting using monoclonal antibodies recognizing glycation adducts, a major glycation adduct, N( epsilon )-(carboxymethyl)lysine was detected. At the late stage of glycation (phase 2), incorporation of AsA reached saturation, and a glycation adduct, pentosidine mediating intramolecular cross-linking, was detected corresponding to formation of high molecular weight aggregates cross-linked between subunits. Glycation led to a decrease in Rubisco activity (half-life about 7-8 d). Furthermore, glycated Rubisco of phase 2 drastically increased protease susceptibility in contrast to unchanged susceptibility of glycated Rubisco of phase 1 compared to that of native Rubisco. Results obtained here suggest that AsA is possibly an important factor in the loss of activity and turnover of Rubisco.