Purification and characterization of leucine aminopeptidase from kidney bean cotyledons

Abstract

Mikkonen, A. 1992. Purification and characterization of leucine aminopeptidase from kidney bean cotyledons. -Physiol. Plant. 84: 393-398.A leucine aminopeptidase (EC 3.4.11.1) was purified from cotyledons of resting kidney beans {Phaseolus vulgaris L. cv. Processor) by acidic extraction, ammonium sulfate fractionation and chromatography on DEAE-Sephacel, Sephacryl S-300, Mono Q HPLC and Superose HPLC columns. The yield of the 317-fold purified enzyme was 9%. On gel filtrations on Sephacryl S-300 and Superose HPLC the elution volumes of the enzyme corresponded to an M^ of 360 000. The enzyme gave one band on native gel electrophoresis and an eiectrophoretic titration in an immobilized pH gradient gave a single curve with a pi of 4.8. Two bands were observed in an SDS-gel electrophoresis with M, values of 58 000 and 60 000 both with and without reduction by 2-mercaptoethanol, indicating that subunits of the enzyme are not linked by disutphide bridges. The purified enzyme most rapidly liberated Leu and Ala ofthe N-termini of di-and oligopeptides, optimally at pH 9.0 ± 0.5. The etizvme was stable in the presence of glycerol, dithiothreitoi and Mg^^, while the latter also had an activating effect. Bestatin inhibited the enzyme competitively with Leu-Gly-Gly with a Kpvalue of 1.5 nM. These observations indicate that the purified aminopeptidase from the cotyledons of resting kidney beans corresponds to the cytosolic leucine aminopeptidase of mammalian tissues (EC 3.4.11.1). The high enzyme activity observed suggests that this aminopeptidase has an important roie in the production of free amino acids during germination.

Isolation and Characterization of the Neutral Leucine Aminopeptidase (LapN) of Tomato

“…In contrast, LAP activity declines before the mobilization of the majority of proteins from kidney bean cotyledons (Mikkonen, 1986). It has been postulated that the levels of the kidney bean LAP may still be sufficient for continued mobilization of protein reserves (Kolehmainen and Mikola, 1971;Mikkonen, 1992). To date, a correlation of LAP-N protein and activity levels in tomato is lacking because LAP-N activities in tissue extracts have not been detected using in situ gel assays.…”

“…Alternatively, the tomato LAP-N expressed in E. coli may not have been able to assemble into its native quaternary structure due to the absence of its in vivo partner in E. coli. There is evidence in kidney beans and humans that heterohexameric LAPs are also present in eukaryotic cells (Sanderink et al, 1988;Mikkonen, 1992).…”

“…For example, in Arabidopsis, LAP proteins accumulate to similar levels in each vegetative and reproductive organ and do not increase in response to phytohormone or stress treatments (Bartling and Nosek, 1994). There is also biochemical evidence for multimeric LAP enzymatic activities in germinated barley (Hordeum vulgare) seeds (green malt) and in resting kidney bean (Phaseolus vulgaris) cotyledons (Kolehmainen and Mikola, 1971;Sopanen and Mikola, 1975;Mikkonen, 1992).…”

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

“…Reaction times were as follows: A, 0 h; B, 1 h; C, 15 h. The products of the digestion of chromogranin A (EEEEEMAVVPQGLFRG-NH 2 , 5E) with the purified enzyme are labeled 4E, 3E, 2E, 1E, and 0E. The products and their molecular weights were as follows: 5E, EEEEEMAVV-PQGLFRG-NH 2 region of the polypeptide that bears many other acidic amino acids residues. There are acidic amino acid-rich regions in two main storage proteins of soybean, the -conglycinin N-terminal and the C-terminal of the glycinin acidic chain.…”

“…1) In addition, many peptidases in cotyledon tissue that play important roles in the degradation process of storage proteins, have been reported. [2][3][4][5] Aminopeptidases catalyze the sequential removal of amino acids from the unblocked N termini of peptides and proteins. Various aminopeptidases with different substrate specificities are widely distributed in eukaryotes and prokaryotes.…”