Exorphins,
peptides with
opioid activity, have previously been isolated from
pepsin hydrolysates of
alpha-casein [Zioudrou, C., Streaty, R. A., & Klee, W. A. (1979) J. Biol. Chem. 254, 2446-2449]. Analysis of these
peptides shows that they correspond to the sequences 90-96,
Arg-Tyr-Leu-Gly-Tyr-Leu-Glu, and 90-95,
Arg-Tyr-Leu-Gly-Tyr-Leu, of
alpha-casein. These
peptides, as well as two of their analogues
Tyr-Leu-
Gly-Tyr-Leu-Glu (91-96) and
Tyr-Leu-
Gly-Tyr-Leu (91-95), have now been synthesized and characterized. Their
opioid activity was examined by three different bioassays: (a) displacement of D-2-alanyl[tyrosyl-3,5-3H]
enkephalin-(5-L-
methioninamide) and [3H]
dihydromorphine from rat brain membranes; (b)
naloxone-reversible inhibition of
adenylate cyclase in homogenates of
neuroblastoma x
glioma hybrid cells; (c)
naloxone-reversible inhibition of electrically stimulated contractions of the mouse vas deferens. The synthetic
peptide of sequence 90-96 was the most potent
opioid in all three bioassays and its potency was similar to that of the isolated
alpha-casein exorphins. The synthetic
peptides were totally resistant to hydrolysis by
trypsin and homogenates of rat brain membranes, but were partially inactivated by
chymotrypsin and
subtilisin. The difference in
opioid activity of
alpha-casein exorphins may be related to differences in conformational flexibility observed by NMR spectroscopy.