Purification and Characterization of an N-Terminal Acidic Amino Acid-Specific Aminopeptidase from Soybean Cotyledons (Glycine max)

Abstract

A novel enzyme that catalyzes the efficient hydrolysis of Glu-Glu was isolated from soybean cotyledons by ammonium sulfate fractionation and successive column chromatographies of Q-sepharose, Phenyl sepharose, and Superdex 200. The apparent molecular mass of this enzyme was found to be 56 kDa and 510 kDa by SDSpolyacrylamide gel electrophoresis and Superdex 200 HR 10/30 column chromatography respectively. The enzyme had high activity against Glu-p-nitroanilide (pNA) and Asp-pNA, whereas Leu-pNA, Phe-pNA, AlapNA, and Pro-pNA were not hydrolyzed. The synthetic dipeptides Glu-Xxx and Asp-Xxx were hydrolyzed, but Xxx-Glu was not. The digestion of a Glu-rich oligopeptide, chromogranin A (Glu-Glu-Glu-Glu-Glu-Met-AlaVal-Val-Pro-Gln-Gly-Leu-Phe-Arg-Gly-NH 2 ) using this purified enzyme was also investigated. Glutamic acid residues were cleaved one by one from the N-terminus. These observations indicate that the enzyme removes glutamyl or aspartyl residues from N-terminal acidic amino acid-containing peptides. It is thought that it was an N-terminal acidic amino acid-specific aminopeptidase from a plant.

Changes and induction of aminopeptidase activities in response to pathogen infection during germination of pigeonpea (Cajanas cajan) seeds

“…The estimated molecular weights of AP1 and AP2 were ∼97 and 42.8 kDa respectively. These molecular weights of pigeonpea seed APs were similar to that of several plant APs reported (Yamaoka et al, 1994;Arima et al, 2000;Ogiwara et al, 2005;Strelec et al, 2009;Tishinov et al, 2009;Asano et al, 2010). While evaluating substrate specificity of inducible pigeonpea seed AP, it was found that the enzyme was preferentially cleaved LpNA.…”

“…The broad pH optimum is due to the titration of three functional groups in the active enzyme centre. Similar broad pH activity profiles were reported for various purified plant APs (Arima et al, 2000;Ogiwara et al, 2005;Strelec et al, 2009;Asano et al, 2010).…”

“…These enzymes are ubiquitous in nature and their activities have been defined in animals, plants and prokaryotes (McDonald and Barrett, 1986;Rendueles and Wolf, 1988;Walling and Gu, 1996). APs have been well characterized from the germinating and non-germinating seeds of the various plant species such as, barley (Hordeum vulgare) (Sopanen and Mikola, 1975;Ogiwara et al, 2005;Strelec et al, 2009), Japanese apricot (Prunus mume) (Ninomiya et al, 1981), Picea abies (Miiller-Starck and Huttermann, 1981), buckwheat (Fagopyrum esculentum) (Ikeda et al, 1983), jojoba (Simmondsia chinensis) (Johnson and Storey, 1985), Vicia faba (Citharel and Garreau, 1987), Haynaldoticum sardoum (Galleschi and Pellegrini, 1989), kidney bean (Phaseolus vulgaris) (Mikkonen, 1992), mung bean (Vigna radiata) (Yamaoka et al, 1994), Cannabis sativa (Arima et al, 2000), sunflower (Helianthus annuus) (Tishinov et al, 2009), chickpea (Cicer arietinum) (Marinova et al, 2009) and soybean (Glycine max) (Asano et al, 2010). All these studies demonstrated the presence of AP activities in resting seeds and at different stages of the seed germination.…”

Structure, trypsin inhibitor activity and functional properties of germinated soybean protein isolate

“…The changes in the levels of free amino acids during soybean germination were measured by HPLC analysis, indicating that the total contents of free amino acids were increased from 515.78 up to 847.44 mg mL −1 (S3 yogurt) and 880.75 mg mL −1 (S6 yogurt) due to the germination (Yang & Li, ). The increase in free amino acids content was attributed to the hydrolysis of polypeptide chains of storage proteins by endogenic exopeptidases during the germination of soybean seeds (Kubota et al ., ; Asano et al ., ). Soy protein, one of the main components of soybean, plays an important role in the performance of soy products, for example soy yogurt.…”

Chlorophyte aspartyl aminopeptidases: Ancient origins, expanded families, new locations, and secondary functions

“…The Arabidopsis thaliana asparaginase 1 (At5g08100) has isoaspartyl dipeptidase activity [20, 21]. In addition, a multimeric aminopeptidase that hydrolyzes Glu and Asp residues from peptides and β-naphthylamide substrates was identified in soybean cotyledons [22]. The soybean aminopeptidase is three fold more active on substrates with N-terminal Glu residues.…”