Benjamin J. Cook

Isolation, primary structure and synthesis of leucomyosuppressin, an insect neuropeptide that inhibits spontaneous contractions of the cockroach hindgut☆

Abstract 1. A neuropeptide that inhibits spontaneous contractions of the isolated cockroach hindgut was purified from head extracts of the cockroach, Leucophaea maderae . 2. The inability of aminopeptidase M to degrade the peptide and the presence of glutamic acid in the hydrolysate suggested N -terminal blocking by pyroglutamic acid. The N -terminal p Glu was removed enzymatically and the unblocked fragment was sequenced with an automated peptide sequencer. 3. The structure determined ( p Glu-Asp-Val-Asp-His-Val-Phe-Leu-Arg-Phe-NH 2 ) was synthesized and shown to be both chemically and biologically identical with the natural product. 4. Leucomyosuppressin is the first inhibitory neuropeptide isolated and structurally identified from an insect source.

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.

Primary structure and synthesis of a blocked myotropic neuropeptide isolated from the cockroach, Leucophaea maderae☆

A peptide which stimulates the contractile activity of the cockroach hindgut was isolated from head extracts of the cockroach, Leucophaea maderae. The inability of aminopeptidase M to degrade the peptide and the presence of glutamic acid in the hydrolysate suggested N-terminal blocking by pyroglutamic acid. The N-terminal pGlu was removed enzymatically and the unblocked fragment was sequenced with an automated gas-phase peptide sequencer. The structure determined (pGlu-Thr-Ser-Phe-Thr-Pro-Arg-Leu-NH2) was synthesized and shown to be both chemically and biologically identical with the natural product.

Isolation, primary structure and synthesis of two neuropeptides from Leucophaea maderae: members of a new family of cephalomyotropins

Abstract 1. 1. Two peptides which stimulate the contractile activity of the cockroach hindgut were isolated from head extracts of the cockroach, Leucophaea maderae. 2. 2. A series of four high-performance liquid-chromatographie (HPLC) fractionations yielded sufficient quantities of pure peptides for micro amino acid analyses and primary sequence determinations. The sequences obtained for the two peptides were: Asp-Pro-Ala-Phe-Asn-Ser-Trp-Gly-NH2 and Asp-Pro-Gly-Phe-Ser-Ser-Trp-Gly-NH2. The two octapeptides were designated leucokinins I and II (L-I, L-II), respectively. 3. 3. Minimum concentrations of synthetic L-I and L-II required to produce a response from the isolated cockroach hindgut were 2.0 × 10−10 and 1.6 × 10−10M, respectively. These concentrations were virtually identical to the threshold concentrations observed with the naturally occurring peptides.

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.

Isolation, primary structure and synthesis of leucokinins VII and VIII: The final members of this new family of cephalomyotropic peptides isolated from head extracts of leucophaea maderae

Abstract 1. Two additional octapeptides, belonging to the leucokinin family of cephalomyotropins, were isolated from head extracts of the cockroach, Leucophaea maderac. 2. A series of 4 high-performance liquid-chromatographic (HPLC) fractionations yielded sufficient quantities of pure peptides for micro amino acid analysis and primary sequence determination. The sequences obtained for the two peptides were: Asp-Pro-Ala-Phe-Ser-Ser-Trp-Gly-NH2 and Gly-Ala-Ser-Phe-Tyr-Ser-Trp-Gly-NH2. They were designated leucokinins VII (L-VII) and VIII (L-VIII) respectively. 3. The minimum concentrations of synthetic L-VII and L-VIII required to evoke a response from the isolated cockroach hindgut were 1.3 × 10−10 M and 2.8 × 10−11 M respectively. 4. L-VII and L-VIII are the lowest titre peptides of the leucokinin series (0.09 and 0.06 picomoles/head equivalent respectively). These are the final leucokinins isolated from our extracts.

Pharmacological properties of excitatory neuromuscular transmission in the hindgut of the cockroach, Leucophaea maderae☆

Abstract Four biologically active materials have been isolated from hindguts of the cockroaches, Leucophaea maderae and Periplaneta americana . Methods for the isolation and characterization of these substances are described. Two of them have been identified as l -glutamic and l -aspartic acids, and they showed transmitter like-action on a hindgut nerve-muscle preparation. In addition to the amino acids, two other myotropic substances were isolated. Both glutamate and aspartate were found in the viscera and nervous system. The hindgut contained the largest amount of glutamate, and the other tissues in the order of decreasing amounts of this amino acid were: terminal ganglion, foregut, and cercal-proctodaeal nerves. The terminal ganglion and cercal-proctodaeal nerves contained the largest amounts of aspartate, whereas the hindgut and foregut contained less. The hindgut of the cockroach L. maderae , was sensitive to both amino acids at 2 × 10 −6 g/ml. Glutamate at 10 −4 g/ml caused a desensitization of neurally evoked contractions of the hindgut, but aspartate at 10 −3 g/ml failed to affect the same degree of desensitization. Both phenylhydrazine hydrochloride (7 × 10 −5 g/ml) and 5-hydroxytryptamine (8 × 10 −4 g/ml) potentiated the neurally elicited response. Dopamine (8 × 10 −5 g/ml) caused an inhibition of both myogenic and neurogenic activity in the hindgut, and it also suppressed glutamate-induced contractions. A structure-activity relationship was determined for various amino acids and related compounds. The presence of two acidic groups and one amino group were essential for excitatory activity.

Comparative pharmacological properties of muscle function in the foregut and the hindgut of the cockroach Leucophaea maderae

Abstract 1. 5-Hydroxytryptamine (4 × 10−8 M) consistently caused an increase in the frequency and amplitude of spontaneous contractions of the foregut of Leucophaea maderae. The hindgut showed similar responses to this indolalkylamine, but the threshold ranged from 4 × 10−8 M to 4 × 10−6 M. 2. Tyramine (5 × 10−6 M) produced a marked suppression of muscular activity in both organs and served as an antagonist of 5-hydroxytryptamine, glutamate and proctolin. 3. Monosodium glutamate (4 × 10−5 M) generally evoked a large phasic contraction in hindguts. However, glutamate (1 × 10−4 M) had either no effect on foreguts or induced a suppression of contractile activity. 4. Although the pentapeptide proctolin stimulated rhythmic activity in both the foregut and the hindgut, the threshold for activation of the hindgut (3 × 10−11 M) was lower than the foregut (3 × 10−8 M). 5. The results suggests that a dichotomy exists between the neurochemical mechanisms controlling these two organs in L. maderae.

The effect of various biogenic amines on the activity of the foregut of the cockroach, Blaberus giganteus

Abstract A differential transformer displacement transducer recorded the activity of the visceral muscle of Blaberus giganteus (L.) with a high degree of sensitivity. The denervated foregut proved to have myogenic properties with certain specific pacemaker regions. Nevertheless, stimulation of the oesophageal nerve innervating the region of the crop caused a slow type of graded contraction in the longitudinal muscles. Preparations of both the innervated and the denervated foregut were sensitive to 5-hydroxytryptamine (10 −7 M), noradrenaline (10 −6 M), and acetylcholine (5 × 10 −6 M). Also, Factor S, a biogenic amine of uncertain structure found in arthropods, caused an excitatory response in innervated foreguts and an inhibitory response in denervated foreguts. Denervated foreguts had a greater sensitivity to l -glutamic acid than innervated preparations. The neurochemical divergence between the control of visceral and somatic musculature of insects is discussed in relation to the pharmacology of these biogenic amines.

Isolation, primary structure, and synthesis of leucokinins V and VI: Myotropic peptides of Lleucophaea maderae☆

Abstract 1. Two additional octapeptides which stimulate the contractile activity of the cockroach hindgut have been isolated from head extracts of the cockroach, Leucophaea maderae . 2. A series of four high performance liquid-chromatographic (HPLC) fractionations yielded sufficient quantities of pure peptides for micro amino acid analysis and primary sequence determinations. The sequence determined for two peptides were: Gly-Ser-Gly-Phe-Ser-Ser-Trp-Gly-NH 2 and pGlu-Ser-Ser-Phe-His-Ser-Trp-Gly-NH 2 . These octapeptides were designated leucokinins V and VI (L-V, L-VI), respectively. 3. Minimum concentrations of natural L—V and L-VI required to produce a response from the isolated cockroach hindgut were 4.5 × and 4.9 × 10 −11 M, respectively. These concentrations were quite similar to the threshold concentrations observed for the synthetic products.