Mobilization of seed protein reserves.

The mobilization of seed storage proteins upon seed imbibition and germination is a crucial process in the establishment of the seedling. Storage proteins fold compactly, presenting only a few vulnerable regions for initial proteolytic digestion. Evolutionarily related storage proteins have similar three-dimensional structure, and thus tend to be initially cleaved at similar sites. The initial cleavage makes possible subsequent rapid and extensive breakdown catalyzed by endo- and exopeptidases. The proteolytic enzymes that degrade the storage proteins during mobilization identified so far are mostly cysteine proteases, but also include serine, aspartic and metalloproteases. Plants often ensure early initiation of storage protein mobilization by depositing active proteases during seed maturation, in the very compartments where storage proteins are sequestered. Various means are used in such cases to prevent proteolytic attack until after imbibition of the seed with water. This constraint, however, is not always enforced as the dry seeds of some plant species contain proteolytic intermediates as a result of limited proteolysis of some storage proteins. Besides addressing fundamental questions in plant protein metabolism, studies of the mobilization of storage proteins will point out proteolytic events to avoid in large-scale production of cloned products in seeds. Conversely, proteolytic enzymes may be applied toward reduction of food allergens, many of which are seed storage proteins.

New serine carboxypeptidase in mung bean seedling cotyledons.

Two serine carboxypeptidases (EC 3.4.16.5) were purified from mung bean seedling cotyledons. Sequences of tryptic peptides derived from the 42.5 kD enzyme corresponded to the derived amino acid sequence of a sequenced cDNA (GenBank U49382 and U49741). This enzyme exhibited the substrate specificity pattern previously published for mung bean carboxypeptidase I. In comparison, the sequence and substrate specificity data obtained for the 43 kD enzyme were similar but not identical. Both enzymes showed preference for peptide substrates with a large hydrophobic residue at the C-terminus. With regard to the penultimate residue of peptide substrates, the mung bean carboxypeptidase I preferred small aliphatic amino acid residues, while the 43 kD enzyme preferred large hydrophobic ones.

Effect of embryonic axis removal and exogenous calcium on carboxypeptidase I of mung bean seedling cotyledons.

Removal of the embryonic axis prevents the normal decline of carboxypeptidase (Cpase) I in mung bean seedling cotyledons. Cpase I activity and protein, the latter manifested on western blots, almost completely disappear about 24 h before the cotyledon abscises. Of the 3 proteolytic enzyme patterns, only that of Cpase I can be restored by an exogenous supply of 10 mM CaCl2 in the agar growth medium. The calcium effect is dependent on [CaCl2] and is not manifested in the presence of chelators and calcium channel blockers. For detached cotyledons to show the normal low level of Cpase I by the eighth day of growth, calcium had to be supplied during seed imbibition and throughout the entire time from removal of the axis. The difference between detached cotyledons in the absence and presence of calcium was greatest when the cotyledons were detached 4-6 days after seed imbibition. Loss of Cpase I activity and protein can be demonstrated in vitro, with the maximum level of Cpase I-degrading activity measured 4 days after seed imbibition under the same growth conditions used to study the calcium effect. It is sensitive to pepstatin and has a pH optimum of 3, suggesting that this Cpase I-degrading activity is due to an aspartic protease.