IMPORTANCE OF THE PROLINE RESIDUE TO THE FUNCTIONAL-ACTIVITY AND METABOLIC STABILITY OF THE NEMATODE FMRFAMIDE-RELATED PEPTIDE, KPNFIRFAMIDE (PF4)

PF4 has previously been shown to have potent inhibitory effects on myo activity of somatic muscle strips from the nematode, Ascaris suum. Thi s study examined the bioactivity and metabolic stability of position 2 - and position 5-modified analogues of PF4. Although the analogues [Le u(5)] PF4, [Ala(2)]PF4, [Gly(2)]PF4, [Ala(2),Leu(5)]PF4, and [Gly(2),L eu(5)]PF4 all had qualitatively similar inhibitory effects on A. suum somatic muscle strips, their effects were quantitatively distinguishab le and had the order of potency: PF4 = [Leu(5)] PF4 >> [Ala(2)]PF4 = [ Ala(2),Leu(5)] PF4 >> [Gly(2)] PF4 = [Gly(2),Leu(5)] PF4. Leu(5) for I le(5) substitutions in PF4 did not alter the activity of this peptide; however, Gly(2)/Ala(2) for Pro(2) substitutions reduced, but did not abolish, peptide activity. Peptide stability studies revealed that [Gl y(2)]PF4(2-7) and -(3-7) and [Ala(2)]PF4(2-7), -(3-7), and -(4-7) frag ments were generated following exposure to A. suum somatic muscle stri ps. However, the parent peptide (PF4) was not metabolized and appeared to be resistant to the sequential cleavages of native aminopeptidases . Observed analogue metabolism appeared to be due to the activity of r eleased aminopeptidases as identical fragments were generated by incub ation in medium that had been exposed to somatic muscle strips and fro m which the strips had been removed prior to peptide addition. It was found that the muscle stretching and bath mixing characteristics of th e tension assay led to more effective release of soluble enzymes from muscle strips and thus greater peptide degradation. These studies reve al that Pro(2) in PF4 is not essential for the biological activity of this peptide; however, it does render the peptide resistant to the act ions of native nematode aminopeptidases. Copyright (C) 1996 Elsevier S cience Inc.