We found that the sequences YPLDL and YPLDLF in the large subunit of spinach D-ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) met the structure YP-aliphatic amino acid which might have opioid activity. We then synthesized these peptides to test their opioid activity. The IC 50 of these peptides in mouse vas deferens assay were 51.0 W WM and 24.4 W WM, respectively, and those in N N receptor binding assay using [ 3 H]deltorphin II as radioligand were 2.09 W WM and 0.93 W WM, respectively. Both peptides were selective for N N receptor. We named them rubiscolin-5 and -6, respectively. Rubiscolin-5 and -6 have antinociceptive activity in mice after i.c.v. or oral administration. The enzymatic conditions to release rubiscolin were investigated using both spinach Rubisco and synthetic fragment peptides. This is the first example of bioactive peptides derived from plant Rubisco. ß
The potent anti-hypertensive peptide, RPLKPW, has been designed based on the structure of ovokinin(2^7). The sequence encoding this peptide was introduced into three homologous sites in the gene for soybean L L-conglycinin K KP P subunit. The native K KP P subunit as well as the modified, RPLKPW-containing K KP P subunit were expressed in Escherichia coli, recovered from the soluble fraction and then purified by ion-exchange chromatography. The RPLKPW peptide was released from recombinant RPLKPW-containing K KP P subunit after in vitro digestion by trypsin and chymotrypsin. Moreover, the undigested RPLKPW-containing K KP P subunit given orally at a dose of 10 mg/kg exerted an anti-hypertensive effect in spontaneously hypertensive rats, unlike the native K KP P subunit. These results provide evidence for the first time that a physiologically active peptide introduced into a food protein by site-directed mutagenesis could practically function in vivo even at a low dose. ß 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.
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