engleski

Aminopeptidases of germinated and non-germinated barley grains

Barley germination is a complex process involving numerous cellular events and wide variety of different enzymes, among which peptidases play important role. Peptidases hydrolyse (a) damaged proteins in cytosol and storage proteins located within storage vacuoles of rehydrated grain living tissues, as well as (b) storage proteins and peptide inhibitors within endosperm of germinating grain. Thus the embryo is supplied with sufficient amount of amino acids necessary for biosynthesis of novel proteins and subsequent development of a new barley plant. According to literature data, at least 42 different endopeptidases and 5 different carboxypeptidases are involved in barley germination, while the exact number of aminopeptidases (AP’ s) and their physiological role still remain to be solved. In order to determine the number of aminopeptidases, induced and involved in seed germination, and to elucidate their possible role in germination process, we have examined AP activities in extracts of barley grains at the beginning of germination (0 h), during germination phases (4, 12, 24 and 72 hour), and in tissues of germinated barley: roots, seedlings and primary leaves. AP activities were determined by colorimetric method using amino acid-2-naphthylamides (2-NA) as substrates and Fast Blue B salt as hydrolysis product coupler. Molecular masses of AP’ s in barley extracts were estimated by gel filtration on Sephacryl S-200, and pI of the most active AP’ s were determined by IEF in pH gradient of 4.5 – 6.0. Additionally, pH optimum of AP’ s in barley extracts was determined. The obtained results indicate that at least 5-6 AP’ s of different substrate specificities, optimally active at pH 7.2-7.6, are present in barley. Among their activities dominated hydrolysis of naphthylamides of hydrophobic amino acids Phe, Leu and Met and basic amino acid Arg, whereas activities towards other substrates were much lower, decreasing in the following order: Ala-, Gly-, Pro- and His-2-NA. AP’ s were found in extracts of dry grains, their activities significantly increasing during the first 24 hours of germination. In developing tissues of germinating grain, seedlings and leaves, AP activities were even higher, and their ratio has changed. Increase of AP activities during the first 24 hours of germination, and their high activities in developing tissues indicate significant role of AP’ s in seed primary metabolism restoration, and in tissue development and growth.

aminopeptidase activities ; barley ; germination ; gel filtration ; IEF

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