G. Mark Holman

Locustatachykinin I and II, two novel insect neuropeptides with homology to peptides of the vertebrate tachykinin family

Two myotropic peptides termed locustatachykinin I (Gly‐Pro‐Ser‐Gly‐Phe‐Tyr‐Gly‐Val‐Arg‐NH2) and locustatachykinin II (Ala‐Pro‐Leu‐Ser‐Gly‐Phe‐Tyr‐Gly‐Val‐Arg‐NH2) were isolated from brain‐corpora cardiaca‐corpora allata‐suboesophageal ganglion extracts of the locust, Locusta migratoria. Both peptides exhibit sequence homologies with the vertebrate tachykinins. Sequence homology is greater with the fish and amphibian tachykinins (up to 45%) than with the mammalian tachykinins. In addition, the intestinal myotropic activity of the locustatachykinins is analogous to that of vertebrate tachykinins. The peptides discovered in this study may just be the first in a whole series of substances from arthropod species to be identified as tachykinin family peptides. Moreover, both chemical and biological similarities of vertebrate and insect tachykinins substantiate the evidence for a long evolutionary history of the tachykinin peptide family.

Leucosulfakinin-II, a blocked sulfated insect neuropeptide with homology to cholecystokinin and gastrin

A sulfated neuropeptide [pGlu-Ser-Asp-Asp-Tyr(SO3H)-Gly-His-Met-Arg-Phe-NH2], with a blocked N-terminus and related to the undecapeptide leucosulfakinin, has been isolated from head extracts of the cockroach, Leucophaea maderae. It exhibits sequence homology with the hormonally-active portion of vertebrate hormones cholecystokinin, human gastrin II and caerulin. This peptide, termed leucosulfakinin-II, shares a common C-terminal heptapeptide fragment with leucosulfakinin and a comparison of the two sequences provides an assessment of the importance of the constituent amino acids to biological activity. Leucosulfakinin-II shows a greater resemblance to cholecystokinin than does leucosulfakinin. Leucosulfakinin-II and leucosulfakinin are the only two reported invertebrate sulfated neuropeptides. As with leucosulfakinin, the intestinal myotropic activity of leucosulfakinin-II is analogous to that of gastrin and cholecystokinin. The sequence homology between the leucosulfakinins and the vertebrate hormones, as well as their analogous myotropic activity, suggest that gastrin/cholecystokinin-like neuropeptides are not confined to vertebrates, but also occur in invertebrates.

Isolation, identification and synthesis of locustamyoinhibiting peptide (LOM-MIP), a novel biologically active neuropeptide from Locusta migratoria.

A novel peptide termed locustamyoinhibiting peptide (LOM-MIP) was isolated from brain-corpora cardiaca-corpora allata-suboesophageal ganglion extracts of the locust, Locusta migratoria. The primary structure of this nonapeptide has been determined Ala-Trp-Gln-Asp-Leu-Asn-Ala-Gly-Trp-NH2. LOM-MIP suppresses the spontaneous contractions of the hindgut and oviduct of Locusta migratoria and of the hindgut of Leucophaea maderae. This novel peptide is, however, structurally different from leucomyosuppressin, a hindgut suppressing peptide isolated from Leucophaea maderae heads. LOM-MIP has a Gly-TrpNH2 carboxy-terminal in common with APGWamide, a penis retractor muscle inhibiting peptide isolated from the snail, Lymnea stagnalis. In addition, it shows carboxy-terminal sequence similarities with locust AKH II which ends in AGWamide. No sequence similarities were found with other vertebrate or invertebrate peptides. Synthetic LOM-MIP showed biological as well as chemical characteristics indistinguishable from those of native LOM-MIP.

The diuretic activity of a series of cephalomyotropic neuropeptides, the achetakinins, on isolated Malpighian tubules of the house cricket, Acheta domesticus

Abstract At 10−9 M achetakinins double the rate of fluid secretion by isolated Malpighian tubules of the house cricket, Acheta domesticus. They appear to act independently of cAMP, and the diuretic responses to a cAMP analogue (8-bromo-cAMP) and achetakinin-I are additive. At 10−9 M achetakinin-I potentiates the diuretic response to exogenous 8-bromo-cAMP. In the presence of 10−5 M 8-bromo-cAMP, achetakinins, I, II, III, IV and V stimulate fluid secretion maximally, and have ED50 values ranging from 1.8 × 10−11 M to 3.2× 10−10 M. The pentapeptide C-terminal sequence FX1X2WG. NH2 (where X1 = H, N, S, Y, and X2 = S or P), common to achetakinins I–V, is all that is required for full biological activity. A possible role for achetakinins as diuretic hormones in vivo is discussed.

Active fragments and analogs of the insect neuropeptide leucopyrokinin: structure-function studies.

Evaluation of analogs of the blocked insect myotropic neuropeptide leucopyrokinin (LPK) has demonstrated its relative insensitivity to amino acid substitution in the N-terminal in contrast to the C-terminal region. Truncated analogs of LPK without the first, second, and third N-terminal amino acids retain a significant 144%, 59% and 30% of the activity of the parent octapeptide, respectively. The [2-8]LPK analog is the first fragment of an insect neuropeptide to exhibit greater activity than the parent hormone. In contrast, truncated analogs of the insect myotropic, proctolin, exhibit little or no activity. The pentapeptide fragment Phe-Thr-Pro-Arg-Leu-NH2 has been identified as the active core of LPK.

Locustatachykinin III and IV : two additional insect neuropeptides with homology to peptides of the vertebrate tachykinin family

Two myotropic peptides termed locustatachykinin III and IV were isolated from 9000 brain-corpora cardiaca-corpora allata-suboesophageal ganglion extracts of the locust, Locusta migratoria. The primary structures of Lom-TK III and IV were established as amidated decapeptides: Ala-Pro-Gln-Ala-Gly-Phe-Tyr-Gly-Val-Arg-NH2 (Lom-TK III) and Ala-Pro-Ser-Leu-Gly-Phe-His-Gly-Val-Arg-NH2 (Lom-TK IV). The locustatachykinins were synthesized and shown to have chromatographic and biological properties identical with those of the native materials. They stimulate visceral muscle contractions of the oviduct and the foregut of Locusta migratoria and of the hindgut of Leucophaea maderae. Both peptides exhibit sequence homologies with the vertebrate tachykinins. Sequence similarity is greater with the fish and amphibian tachykinins (up to 40%) than with the mammalian tachykinins. In addition, the intestinal and oviducal myotropic activity of the locustatachykinins is analogous to that of vertebrate tachykinins. Both chemical and biological similarities of vertebrate and insect tachykinins substantiates the evidence for a long evolutionary history of the tachykinin peptide family.

Isolation, identification and synthesis of PDVDHFLRFamide (SchistoFLRFamide) in Locusta migratoria and its association with the male accessory glands, the salivary glands, the heart, and the oviduct

An amidated decapeptide, exhibiting strong inhibitory activity of spontaneous visceral muscle movements, was isolated from 9000 brain-corpora cardiaca-corpora allata-subesophageal ganglion complexes of the migratory locust, Locusta migratoria. During the process of HPLC purifications, the biological activity of the fractions was monitored using the isolated hindgut of the cockroach Leucophaea maderae. The primary structure of this myotropic peptide is Pro-Asp-Val-Asp-His-Val-Phe-Leu-Arg-Phe-NH2 and is identical to SchistoFLRFamide isolated from the grasshopper, Schistocerca gregaria. It shares the carboxy-terminal sequence FLRFamide with several identified peptides from different phyla. At this moment, six decapeptides isolated from different insect species are identical at 7 of the 10 amino acid residues (X-D-V-X-H-X-FLRFamide). The cockroach, fly, and locust peptides differ only by the N-terminal amino acid residue. Synthetic SchistoFLRFamide showed biological as well as chemical characteristics indistinguishable from the native peptide. It provoked a decrease in frequency and amplitude of contractions of the locust oviduct. By means of a polyclonal antiserum directed against the carboxy terminal of SchistoFLRFamide, we demonstrated that the male accessory glands, the heart, the oviduct, and the salivary glands were innervated by axons containing SchistoFLRFamide-like immunoreactivity. Administration of SchistoFLRFamide elicited an immediate effect on the basal membrane potential of the opalescent tubule gland cells.

Isolation, primary structure, and synthesis of locustapyrokinin: a myotropic peptide of Locusta migratoria.

A neuropeptide which stimulates the motility of the cockroach hindgut has been isolated from an extract of 9000 brain-corpora cardiaca-corpora allata-subesophageal ganglion complexes of Locusta migratoria. Biological activity was monitored during HPLC purification by observing the myotropic effect of column fractions on the isolated hindgut of Leucophaea maderae. The primary structure of this myotropic peptide was established as a blocked 16-residue peptide: pGlu-Asp-Ser-Gly-Asp-Gly-Trp-Pro-Gln-Gln-Pro-Phe-Val-Pro-Arg-Leu-NH2. This novel locust peptide was designated as locustapyrokinin, or Lom-PK. Lom-PK was synthesized and shown to have chromatographic and biological properties identical to those of the native material. Lom-PK has a Phe-X-Pro-Arg-Leu-NH2 carboxy terminal in common with leucopyrokinin (or Lem-PK), a blocked myotropic neuropeptide isolated from the cockroach hindgut. The constituent amino acids of this C-terminal are important for biological activity on the Leucophaea hindgut. The primary structure of this novel insect peptide is, however, substantially different from Lem-PK at the amino-terminal sequence.

Locustakinin, a novel myotropic peptide from Locusta migratoria, isolation, primary structure and synthesis.

The isolated hindgut of the cockroach, Leucophaea maderae is a very efficient bioassay tool for the monitoring of certain structural types of insect myotropic peptides during HPLC purification. Using this detection system, a six residue peptide has been isolated from an extract of 9000 brain corpora cardiaca-corpora allata suboesophageal ganglion complexes of Locusta migratoria. Amino acid composition and sequence analysis combined with enzymatic digestion data established the structure of the novel peptide as Ala-Phe-Ser-Ser-Trp-Gly-amide. The chromatographic and biological properties of the synthetic peptide were the same as those of the native peptide, thus confirming structural analysis. The carboxy-terminal pentamer sequence is the active core of leucokinins II, V and VII and of achetakinin III (myotropic neuropeptides isolated from Leucophaea m. and from Acheta domesticus; Holman et al., 1990). Furthermore, the octapeptide leucokinin VII contains the novel sequence as its carboxy-terminal hexamer and Achetakinin V (AFHSWGamide) differs from it by one residue. This new peptide designated as locustakinin I (locusts) may therefore represent an evolutionary molecular link between leucokinin VII (cockroaches) and achetakinin V (crickets). Using synthetic locustakinin, physiological studies will be performed in the locust. In view of the known effects of leucokinins, locustakinin may be important in the stimulation of ion transport and inhibition of diuretic activity in Malpighian tubules. This study indicates that the AFXSWGamide sequence appears to have been well conserved and that members of this peptide family may be widely distributed among insects and posses a number of functions.

Isolation and primary structure of a peptide from the corpora cardiaca of Heliothis zea with adipokinetic activity

An adipokinetic hormone was isolated from the corpora cardiaca of the corn ear worm moth, Heliothis zea, and purified by reversed phase high performance liquid chromatography. The primary structure, pGlu-Leu-Thr-Phe-Thr-Ser-Ser-Trp-Gly-NH2, was determined by automated gas-phase Edman degradation of the peptide deblocked with pyroglutamic aminopeptidase, and by fast atom bombardment mass spectrometry. The hormone was synthesized and the natural and synthetic material had identical chromatographic, spectroscopic, and biological properties. The peptide was found to have lipid mobilizing activity in H. zea adults.