Allison Strey

Culekinin depolarizing peptide: a mosquito leucokinin-like peptide that influences insect Malpighian tubule ion transport

A peptide termed culekinin depolarizing peptide (CDP) was isolated from approximately 1.2 million mosquitos (94% Culex salinarius). The peptide was isolated on the basis of a rapid myotropic assay that utilized a hindgut preparation from Leucophaea maderae and a transepithelial voltage assay that used mosquito Malpighian tubules from Aedes aegypti. A 15% trifluoroacetic acid extraction from the mosquitos, two solid phase extraction steps, and six HPLC steps resulted in the isolation of 9.7 nmol of CDP. This value corresponds to approximately 8 fmol/mosquito. Edman degradation indicated the following sequence for CDP: Asn-Pro-Phe-His-Ser-Trp-Gly-NH2. The sequence was confirmed as the suspected C-terminal amide form of the peptide, since native and synthetic CDP had identical chemical and biological properties. CDP is a member of the leucokinin family of neuropeptides. The leucokinins have been found in three other insect species (Leucophaea maderae, Acheta domesticus and Locusta migratoria) where these peptides were isolated by their myotropic properties alone. CDP shares a C-terminal sequence homology (i.e., Phe-X-Ser-Trp-Gly-NH2) with the rest of the leucokinins. CDP corresponds to the strongest tubule depolarizing activity in the C. salinarius extract. These findings agree with previous structure-activity studies that suggest that mosquitos would contain a leucokinin-like factor that had Phe-His-Ser-Trp-Gly-NH2 as the C-terminal pentapeptide. This is the first leucokinin isolated from blood feeding or holometabolous insects.

Structure-activity studies of allatostatin 4 on the inhibition of juvenile hormone biosynthesis by corpora allata: The importance of individual side chains and stereochemistry

The production of juvenile hormone III (JH III) by the corpora allata of the cockroach Diploptera punctata is regulated in part by peptides originating from the brain. One group of these peptides, termed allatostatins, reversibly inhibits the biosynthesis of JH in vitro. Allatostatin 4 (AST4: Asp-Arg-Leu-Tyr-Ser-Phe-Gly-Leu-amide) is the smallest member of the AST family yet defined and was used as the benchmark peptide for these initial structure-activity studies. Two initial analog series of AST4 were examined for the ability of each analog to inhibit JH biosynthesis by corpora allata in vitro. Each analog series consisted of analogs that contained a single amino acid change from the native AST4 sequence. The first series contained Ala replacement analogs and the second contained analogs with D-amino acid replacements. The first analog series used Ala replacements to help indicate which amino acid side chains were most important for inhibition of JH biosynthesis. The most important side chain appeared to be Leu8 followed by Phe6 and Tyr4. Additionally, the D-amino acid series suggested that a secondary structural element(s) at the C-terminus of AST4 could be important to the biological activity.

Hydrolysis of Insect Neuropeptides by an Angiotensin-Converting Enzyme From the Housefly, Musca domestica

The presence in insect tissues of peptides with structural similarities to angiotensin I and to bradykinin, the two best known substrates of mammalian angiotensin-converting enzyme, has not been reported. As part of our study to identify potential substrates for insect angiotensin-converting enzyme, we have investigated the susceptibility of a number of known insect peptide hormones and neurotransmitters to hydrolysis by Musca domestica angiotensin-converting enzyme. Insect peptides belonging to the red pigment-concentrating hormone, leucokinin, locust tachykinin, and depolarizing peptide families were hydrolyzed by housefly angiotensin-converting enzyme, whereas proctolin and crustacean cardioactive peptide were not substrates. Cus-DP II, LK I, LK II, and Lom-TK I were all cleaved at the penultimate C-terminal peptide bond to release a dipeptide amide as a major fragment with Km values of 94 +/- 11, 634 +/- 8, and 296 +/- 35 microM for Cus-DP II, LK I, and Lom-TK I, respectively. The ability of insect angiotensin-converting enzyme to hydrolyze C-terminally amidated peptides in vitro might be of functional significance because the enzyme has been localized to neuropile regions of the insect brain and is present in the hemolymph of houseflies.

Identification and partial characterization of receptors for allatostatins in brain and corpora allata of the cockroach Diploptera punctata using a binding assay and photoaffinity labeling

We have developed both an in vitro binding assay and a photoaffinity labeling assay to demonstrate and partially characterize putative receptors for allatostatins in brain and in corpora allata of Diploptera punctata. Isolated brain membranes were photoaffinity labeled with 125I-RYBPA (photoaffinity analogue of dip-allatostatin 5). Following labeling with 125I-RYBPA, SDS-PAGE and autoradiography revealed the presence of a putative receptor (37 kDa) for dip-allatostatin 5 and dip-allatostatin 7. Specific labeling was demonstrated by dose-dependent competition with either dip-allatostatin 5 or dip-allatostatin 7. The in vitro binding assay indicated that the receptor for dip-allatostatin 5 had a Kd of (9.0 +/- 0.9).10(-10) M and Bmax of 2.2 +/- 0.3 pmol/mg membrane protein. For dip-allatostatin 7, two Kd values of (1.5 +/- 0.1).10(-9) M and (3.8 +/- 0.3).10(-9) M were obtained, with Bmax values of 7.2 +/- 0.7 pmol/mg membrane protein and 11.4 +/- 1.0 pmol/mg membrane protein respectively. This indicates that there were probably two putative receptor sites for dip-allatostatin 7 although only one band was observable following photoaffinity labeling. Binding was saturable, specific and reversible. Using the in vitro binding assay, the Kd of the putative receptor in CA for dip-allatostatin 7 was shown to be (7.2 +/- 0.9).10(-10) M.

Characterization of a leucokinin binding protein in Aedes aegypti (Diptera: Culicidae) Malpighian tubule

The insect myokinin (leucokinin-like) neuropeptide family includes peptides that have different physiological effects such as the induction of hindgut myotropic activity and stimulation of urine production. The C-terminal pentamer of myokinins Phe-X-(Ser/Pro/Ala)-Trp-Gly-amide [X=Phe, His, Asn, Ser or Tyr], had been previously determined as the minimum fragment able to elicit a functional response. The receptor(s) for these insect neuropeptides has not yet been identified. In order to characterize the Malpighian tubule leucokinin-like peptide receptor(s) from the yellow fever mosquito (Aedes aegypti), a leucokinin photoaffinity analogue (LPA) of sequence dAla-dTyr-Bpa-dLys-Phe-Phe-Ser-Trp-Gly-amide was designed based on structure/activity relationships for leucokinins. LPA caused depolarization of the transepithelial voltage (TEV) in female Malpighian tubule, confirming the activity of the peptide. The effective concentration to give half the maximum depolarization (EC(50)) was 17 nM. The (125)I-LPA was then used to characterize leucokinin binding proteins in female Malpighian tubule membranes. It specifically labeled and saturated a protein(s) of about 54 kDa as shown by SDS-PAGE/autoradiography and by competition experiments with excess unlabeled leucokinin analogues. (125)I-LPA bound to the 54 kDa protein(s) with a K(d) value of 13+/-3 nM in agreement with the EC(50) for the TEV bioassay. Altogether these data suggest that the 54 kDa protein is an Aedes-leucokinin receptor. This is the first characterization of an insect leucokinin receptor and reveals that LPA is a powerful tool to label insect myokinin receptors.

Adsorption Chromatography of Small C-Terminal Peptide Amides on Dihydroxyalkyl Bonded Silica High Performance Liquid Chromatography Columns and Application to Purification of Insect Neuropeptides

The chromatography of small neuropeptides (< 1400 mw) was studied on a high performance liquid chromatography (HPLC) sized exclusion column designed with an aqueous-compatible dihydroxyalkyl bonded silica support. The major mechanism of retention for small peptide amides on this column was clearly not size exclusion but adsorption. Peptide amides were nearly always retained beyond the calculated salt volume of these columns. This retention was nearly five column volumes for selected peptides like substance P. Under the same conditions, peptide C-terminal acid analogs always eluted much closer to retention times predicted by their molecular weight. Changes in the composition of the mobile phase indicated that the selective retention of the peptide amides was primarily the result of selective yet weak hydrophobic interactions. However, most peptide amides will elute from these supports without the aid of organic solvents, an ideal characteristic for direct coupling of this method to reversed-phase HPLC. A pilot two-dimensional (dihydroxyalkyl HPLC/reversed-phase HPLC) HPLC purification of a diuretic peptide from mosquito head extracts showed that the coupling of these two forms of chromatography could be used to quickly isolate a minor component of a complex mixture.