Quantitation and sensory properties of three newly identified pyroglutamyl oligopeptides in sake

Three new peptides: (pGlu)L-ethyl, (pGlu)LFGP-ethyl and (pGlu)LFNP-ethyl, were identified in the search for pyroglutamyl oligopeptide ethyl esters in sake. The ethyl esterified peptides in sake were quantitated using stable isotope dilution analysis and additional quantitation of (pGlu)L was performed using an external standard method. The concentrations of (pGlu)L-ethyl and (pGlu)L in 33 commercial sake samples ranged from 0.16 to 1.57 mg/L and 1.49 to 7.55 mg/L, respectively. The sensory properties of the pyroglutamyl oligopeptide ethyl esters and corresponding non-esterified peptides were examined: the estimated difference threshold of (pGlu)L (2.0 mg/L) and (pGlu)L-ethyl (0.267 mg/L) was exceeded in 32 and 26 samples, respectively. Estimated thresholds of (pGlu)LFGP-ethyl and (pGlu)LFNP-ethyl were often lower than the levels in quantitated sake samples. The sensory effects of these pyroglutamyl dipeptides on a model sake quality may be negative because of their unpleasant taste, however, (pGlu)LFNP-ethyl may be positive because of its mild taste.     

On the seeding and oligomerization of pGlu-amyloid peptides (in vitro)

Oligomerization of amyloid beta (Abeta) peptides is the decisive event in the development of Alzheimer's disease (AD), the most common neurogenerative disorder in developed countries. Recent evidence links this conformation-driven process to primary- and secondary-structure modifications of Abeta. The N and C terminus of deposited Abeta has been shown to possess conspicuous heterogeneity. While the C-terminally longer form of Abeta, i.e., Abeta (42), is considered more amyloidogenic, the role of the N-terminal modifications, e.g., truncation and glutamate cyclization accounting for the majority of the deposited peptides, is less understood. In the present study, we characterized the oligomerization and seeding capacity of pGlu-amyloid peptides using two unrelated techniques based on flow cytometry or flourescence dye binding. Under different conditions and irrespective of the C terminus of Abeta, i.e., Abeta40 or 42, pGlu-modified peptides displayed an up to 250-fold accelerated initial formation of aggregates compared to unmodified Abeta. The accelerated seed formation is accompanied by a change in the oligomerization kinetics because of N-terminal pGlu formation. Furthermore, the formation of mixed aggregates consisting of either pGlu-Abeta (3-42) or ADan or ABri and Abeta (1-42) was investigated by Abeta fluorescence labeling in flow cytometry. The results suggest that pGlu-modified peptides are potential seeding species of aggregate formation in vivo. The data presented here and the abundance of pGlu peptides in amyloidoses, such as FBD and AD, suggest pGlu-amyloid peptides as a species with biophysical characteristics that might be in particular crucial for the initiation of the disease.     

Growth cones isolated from identified Aplysia neurons in vitro: biochemical and morphological characterization

The right upper quadrant (RUQ) cells (R3-R13) of Aplysia regenerating in dissociated cell culture form unusually large growth cones. The movement of these growth cones was observed by time-lapse phase microscopy and their ultrastructure was examined by transmission electron microscopy. Their behavior and ultrastructure have features that are typical of growth cones in vitro. Additionally, they contain neurosecretory granules similar to those found in these cells in vivo. Because RUQ growth cones are large, they can be isolated by manual dissection. RUQ cells were grown in the presence of [35S]methionine and the labeled proteins transported to the growth cones were analyzed by SDS-PAGE. These proteins were compared to those in RUQ cell bodies, RUQ neurites, and to those in the neurites and cell bodies of other identified neurons grown in vitro. Most proteins synthesized by RUQ cells in vitro are transported to their growth cones, including several glycoproteins and the precursor to the R3-R14 neuropeptide. Neuropeptides are also synthesized by a number of other Aplysia neurons growing in vitro. We examined R2, LPL1, R15, and left upper quadrant neurons and found that their precursor peptides, like those of R3-R14, are readily recognized as major cell-specific radiolabeled bands on SDS gels. The presence in regenerating growth cones of neuropeptides, neurosecretory granules, and glycoproteins known to be rapidly transported toward synapses in vivo supports the emerging view that the growth cone in vitro contains not only a motility apparatus but also a macromolecular assembly capable of forming an active synapse immediately upon or shortly after contacting targets.     

Amino acid uptake and incorporation not cell-specific peptides and evidence for intracellular peptide pools in Aplysia neurons R3-R14

1. Relationships between intracellular amino acid concentrations and uptake rates and their utilization in synthesis of cell-specific peptides in neurons R3-R14 in the Aplysia parietovisceral ganglion are explored. 2. The uptake rates and intracellular concentrations of most amino acids are positively correlated and inversely related to their degree of incorporation into the peptides. 3. The bulk cellular pool of arginine is probably utilized in the synthesis of R3-R14 peptides, but much of the glycine taken up appears not to be readily available for protein synthesis. 4. There are rapidly and slowly turning over pools of the peptides, and portions of the peptides stay in the cell bodies for days.     

Inhibition of glutaminyl cyclase alters pyroglutamate formation in mammalian cells

Mammalian cell lines were examined concerning their Glutaminyl Cyclase (QC) activity using a HPLC method. The enzyme activity was suppressed by a QC specific inhibitor in all homogenates. Aim of the study was to prove whether inhibition of QC modifies the posttranslational maturation of N-glutamine and N-glutamate peptide substrates. Therefore, the impact of QC-inhibition on amino-terminal pyroglutamate (pGlu) formation of the modified amyloid peptides Abeta(N3E-42) and Abeta(N3Q-42) was investigated. These amyloid-beta peptides were expressed as fusion proteins with either the pre-pro sequence of TRH, to be released by a prohormone convertase, or as engineered amyloid precursor protein for subsequent liberation of Abeta(N3Q-42) after beta- and gamma-secretase cleavage during posttranslational processing. Inhibition of QC leads in both expression systems to significantly reduced pGlu-formation of differently processed Abeta-peptides. This reveals the importance of QC-activity during cellular maturation of pGlu-containing peptides. Thus, QC-inhibition should impact bioactivity, stability or even toxicity of pyroglutamyl peptides preventing glutamine and glutamate cyclization.     

Effects of synthetic bag cell and atrial gland peptides on identified nerve cells in Aplysia

We have examined the effects of peptides on the neuroendocrine bag cells, the R2 neuron and the left upper quadrant (LUQ) neurons of the abdominal ganglion of Aplysia californica. Peptides include those extracted from the atrial gland, a reproductive organ; those released by an afterdischarge of the bag cells; and 2 synthetic peptides: the amidated 9-amino acid C-terminal portion of atrial gland peptides A/B/ERH (B26-34), and the 8-amino acid alpha-bag cell peptide (alpha-BCP1-8). Peptides were applied by superfusion, arterial perfusion, pressure ejection from micropipettes, or by inducing a bag cell afterdischarge. Both alpha-BCP1-8 and B26-34 are able to produce a bag cell afterdischarge when applied to the abdominal ganglion but are not as effectively able to trigger the bag cells when applied selectively to the ganglia of the head ring. Peptides released by the bag cells inhibit R2 and LUQ neurons; whereas atrial gland extract mildly excites LUQ neurons and powerfully excites R2. The inhibitory effect of the LUQ cells and R2 following an afterdischarge of the bag cells is mimicked by alpha-BCP1-8. The excitatory effect of the atrial gland extract cannot be duplicated with B26-34. Rather, instead of having an excitatory effect on R2 and LUQ cells, B26-34 seems to mimick alpha-BCP1-8 and inhibit these neurons. Both peptides produce a membrane conductance increase in R2 and LUQ cells.     

N-Terminal pyroglutamate formation of Aβ38 and Aβ40 enforces oligomer formation and potency to disrupt hippocampal long-term potentiation

Pyroglutamate (pGlu)-modified amyloid peptides have been identified in sporadic and familial forms of Alzheimer's disease (AD) and the inherited disorders familial British and Danish Dementia (FBD and FDD). In this study, we characterized the aggregation of amyloid-β protein Aβ37, Aβ38, Aβ40, Aβ42 and ADan species in vitro, which were modified by N-terminal pGlu (pGlu-Aβ3-x, pGlu-ADan) or possess the intact N-terminus (Aβ1-x, ADan). The pGlu-modification confers rapid formation of oligomers and short fibrillar aggregates. In accordance with these observations, the pGlu-modified Aβ38, Αβ40 and Αβ42 species inhibit hippocampal long term potentiation of synaptic response, but pGlu-Aβ3-42 showing the highest effect. Among the unmodified Aβ peptides, only Aβ1-42 exhibites such propensity, which was similar to pGlu-Aβ3-38 and pGlu-Aβ3-40. Likewise, the amyloidogenic peptide pGlu-ADan impaired synaptic potentiation more pronounced than N-terminal unmodified ADan. The results were validated using conditioned media from cultivated HEK293 cells, which express APP variants favoring the formation of Aβ1-x, Aβ3-x or N-truncated pGlu-Aβ3-x species. Hence, we show that the ability of different amyloid peptides to impair synaptic function apparently correlates to their potential to form oligomers as a common mechanism. The pGlu-modification is apparently mediating a higher surface hydrophobicity, as shown by 1-anilinonaphtalene-8-sulfonate fluorescence, which enforces potential to interfere with neuronal physiology.     

14-3-3eta is a novel regulator of parkin ubiquitin ligase

Mutation of the parkin gene, which encodes an E3 ubiquitin-protein ligase, is the major cause of autosomal recessive juvenile parkinsonism (ARJP). Although various substrates for parkin have been identified, the mechanisms that regulate the ubiquitin ligase activity of parkin are poorly understood. Here we report that 14-3-3eta, a chaperone-like protein present abundantly in neurons, could bind to parkin and negatively regulate its ubiquitin ligase activity. Furthermore, 14-3-3eta could bind to the linker region of parkin but not parkin with ARJP-causing R42P, K161N, and T240R mutations. Intriguingly, alpha-synuclein (alpha-SN), another familial Parkinson's disease (PD) gene product, abrogated the 14-3-3eta-induced suppression of parkin activity. alpha-SN could bind tightly to 14-3-3eta and consequently sequester it from the parkin-14-3-3eta complex. PD-causing A30P and A53T mutants of alpha-SN could not bind 14-3-3eta, and failed to activate parkin. Our findings indicate that 14-3-3eta is a regulator that functionally links parkin and alpha-SN. The alpha-SN-positive and 14-3-3eta-negative control of parkin activity sheds new light on the pathophysiological roles of parkin.     

Isolation of high-affinity peptide antagonists of 14-3-3 proteins by phage display.

The 14-3-3 proteins interact with diverse cellular molecules involved in various signal transduction pathways controlling cell proliferation, transformation, and apoptosis. To aid our investigation of the biological function of 14-3-3 proteins, we have set out to identify high-affinity antagonists. By screening phage display libraries, we have identified a set of peptides which bind 14-3-3 proteins. One of these peptides, termed R18, exhibited a high affinity for different isoforms of 14-3-3 with estimated K(D) values of 7-9 x 10(-)(8) M. Recognition of multiple isoforms of 14-3-3 suggests the targeting of R18 to a structure that is common among 14-3-3 proteins, such as the conserved ligand-binding groove. Indeed, mutations that alter critical residues in the ligand-binding site of 14-3-3 drastically decreased the level of 14-3-3-R18 association. R18 efficiently blocked the binding of 14-3-3 to the kinase Raf-1, a physiological ligand of 14-3-3, and effectively abolished the protective role of 14-3-3 against phosphatase-induced inactivation of Raf-1. The cocrystal structure of R18 in complex with 14-3-3zeta revealed the occupancy of the general binding groove of 14-3-3zeta by R18, explaining the potent inhibitory effect of R18 on 14-3-3-ligand interactions. Such a well-defined peptide will be an effective tool for probing the role of 14-3-3 in various signaling pathways, and may lead to the development of 14-3-3 antagonists with pharmacological applications.     

Separate cell types that express two different forms of somatostatin in anglerfish islets can be immunohistochemically differentiated

The somatostatin-related peptides somatostatin-14 (SS-14) and somatostatin-28 (aSS-28) are synthesized at the C-terminal end of two separate pre-pro-somatostatins in anglerfish pancreatic islets. The purpose of this study was to determine whether these peptides are expressed in the same or different cell types. Antisera R141 and R293, which recognize the central region of SS-14 and the C-terminal region of aSS-28 ([Tyr7,Gly10] SS-14), respectively, were used in an immunohistochemical examination of anglerfish islets. The R293 antiserum-labeled cells were distributed individually or in small clusters. These same cells, as well as a separate set of cells arranged in large clusters, were stained by the R141 antiserum. Pre-absorption of the R141 antiserum with [Tyr7,Gly10] SS-14 eliminated staining by R141 of only those cells also labeled by R293, whereas pre-absorption of R141 with SS-14 prevented all staining. Pre-absorption of R293 with [Tyr7,Gly10] SS-14 eliminated all staining, whereas pre-absorption with SS-14 had no effect on aSS-28-like immunoreactivity. These results suggest the existence of two separate cell types which express either SS-14 or aSS-28. The cells that contained the somatostatin-related peptides were found to be distinct from those cells that contained insulin, glucagon, or anglerfish peptide Y. However, the cells stained by the R293 antiserum were distributed in close association with glucagon-containing cells. The implications of the existence of separate cell types which express SS-14 or aSS-28 are discussed with regard to processing of the biosynthetic precursors to these peptides.     

Glutaminyl cyclases from animals and plants: a case of functionally convergent protein evolution

Several mammalian peptide hormones and proteins from plant and animal origin contain an N-terminal pyroglutamic acid (pGlu) residue. Frequently, the moiety is important in exerting biological function in either mediating interaction with receptors or stabilizing against N-terminal degradation. Glutaminyl cyclases (QCs) were isolated from different plants and animals catalyzing pGlu formation. The recent resolution of the 3D structures of Carica papaya and human QCs clearly supports different evolutionary origins of the proteins, which is also reflected by different enzymatic mechanisms. The broad substrate specificity is revealed by the heterogeneity of physiological substrates of plant and animal QCs, including cytokines, matrix proteins and pathogenesis-related proteins. Moreover, recent evidence also suggests human QC as a catalyst of pGlu formation at the N-terminus of amyloid peptides, which contribute to Alzheimer's disease. Obviously, owing to its biophysical properties, the function of pGlu in plant and animal proteins is very similar in terms of stabilizing or mediating protein and peptide structure. It is possible that the requirement for catalysis of pGlu formation under physiological conditions may have triggered separate evolution of QCs in plants and animals.     

Complete crystal structure of monocyte chemotactic protein-2, a CC chemokine that interacts with multiple receptors

Monocyte chemotactic protein 2 (MCP-2) is a CC chemokine that utilizes multiple cellular receptors to attract and activate human leukocytes. MCP-2 is a potent inhibitor of HIV-1 by virtue of its high-affinity binding to the receptor CCR5, one of the major coreceptors for HIV-1. Although a few structures of CC chemokines have been reported, none of these was determined with the N-terminal pyroglutamic acid residue (pGlu1) and a complete C-terminus. pGlu1 is essential for the chemotactic activity of MCP-2. Recombinant MCP-2 has Gln1 at the N terminus, 12-15% of which cyclizes automatically and forms pGlu1. The chemotactic activity of such MCP-2 mixture, which contains 12-15% pGlu1-form and 85-88% Gln1-form protein, is approximately 10 times lower when compared with that of fully cyclized MCP-2 preparation. Therefore, this chemokine is practically inactive without pGlu1. We have determined the complete crystal structure of MCP-2 that contains both pGlu1 and an intact C-terminus. With the existence of pGlu1, the conformation of the N-terminus allows two additional interactions between the two subunits of MCP-2 dimer: a hydrogen bond between pGlu1 and Asn17 and a salt bridge between Asp3 and Arg18. Consequently, both pGlu1 are anchored and buried, and thereby, both N-terminal regions are protected against protease degradation. We have also observed not previously reported extended helical nature of the C terminal region, which covers residues 58-74.     

Inhibition of glutaminyl cyclase attenuates cell migration modulated by monocyte chemoattractant proteins

QC (glutaminyl cyclase) catalyses the formation of N-terminal pGlu (pyroglutamate) in peptides and proteins. pGlu formation in chemoattractants may participate in the regulation of macrophage activation and migration. However, a clear molecular mechanism for the regulation is lacking. The present study examines the role of QC-mediated pGlu formation on MCPs (monocyte chemoattractant proteins) in inflammation. We demonstrated in vitro the pGlu formation on MCPs by QC using MS. A potent QC inhibitor, PBD150, significantly reduced the N-terminal uncyclized-MCP-stimulated monocyte migration, whereas pGlu-containing MCP-induced cell migration was unaffected. QC small interfering RNA revealed a similar inhibitory effect. Lastly, we demonstrated that inhibiting QC can attenuate cell migration by lipopolysaccharide. These results strongly suggest that QC-catalysed N-terminal pGlu formation of MCPs is required for monocyte migration and provide new insights into the role of QC in the inflammation process. Our results also suggest that QC could be a drug target for some inflammatory disorders.     

Achieving specificity in selected and wild-type N peptide-RNA complexes: the importance of discrimination against noncognate RNA targets

The boxB RNA pentaloops from the P22 and lambda phages each adopt a GNRA tetraloop fold upon binding their cognate arginine-rich N peptides. The third loop base in P22 boxB (3-out) and the fourth in lambda boxB (4-out) are excluded to accommodate this structure. Previously, we selected a pool of lambda N sequences with random amino acids at loop contacting positions 13-22 for binding to either of these two GNRA-folded pentaloops or a canonical GNRA tetraloop and isolated a class of peptides with a new conserved arginine (R15). Here, we characterize the binding of lambda N and these R15 peptides using fluorescent titrations with 2-aminopurine labeled versions of the three GNRA-folded loops and circular dichroism spectrometry. All peptides preferentially bind the lambda boxB RNA loop. lambda N and R15 peptide specificity against the P22 loop arises from the cost of rearranging its loop into the 4-out GNRA structure. Modeling indicates that the interaction of R8 with an additional loop phosphate in the 4-out GNRA pentaloop selectively stabilizes this complex relative to the tetraloop. R15 peptides gain additional discrimination against the tetraloop because their arginine also preferentially interacts with the 4-out GNRA pentaloop phosphate backbone, whereas K14 and W18 of lambda N contribute equal affinity when binding the tetraloop. Nonspecific electrostatic interactions by basic residues near the C-termini of these peptides create significantly steeper salt dependencies in association constants for noncognate loops, aiding discrimination at high salt concentrations. Our results emphasize the importance of considering specificity against noncognate as well as nonspecific targets in the combinatorial and rational design of biopolymers capable of macromolecular recognition.     

Differentiated properties of identified serotonin neurons in dissociated cultures of embryonic rat brain stem

Serotonin neurons in 14-d embryonic rat brain stem were identified by peroxidase-antiperoxidase immunocytochemistry with an affinity-purified antiserotonin antibody. Brain-stem tissue was dissected from 14- or 15- d embryonic rats, dissociated and grown in cell culture for up to 5 wk, and serotonin neurons were identified by immunocytochemistry. Within 24 h of plating, serotonin immunoreactivity was present in 3.3% of neurons. Immunoreactivity in neuronal cell bodies decreased with time, whereas staining of processes increased. The number of serotonin- immunoreactive neurons remained constant at 3-5% over the first 14 d in culture. From 14 to 28 d, the total number of neurons decreased with little change in the number of serotonin neurons, such that, by day 28 in culture, up to 36% of surviving neurons exhibited serotonin immunoreactivity. Similar percentages of cultured brain stem neurons accumulating 3H-serotonin were identified by autoradiography. Uptake was abolished by the serotonin-uptake inhibitor, clomipramine, but was unaffected by excess norepinephrine, or by the norepinephrine-uptake inhibitor, maprotiline. Synthesis of 3H-serotonin was detected after incubation of cultures with 3H-tryptophan, and newly synthesized serotonin was released by potassium depolarization in a calcium- dependent manner. More than 95% of serotonin neurons were destroyed after incubation of cultures with 5,6-dihydroxytryptamine. Brain-stem cultures contained virtually no neurons with the ability to accumulate 3H-norepinephrine or 3H-dopamine. Approximately 40% of brain-stem neurons were labeled with gamma-aminobutyric acid (3H-GABA). However, there was almost no overlap in the surface area of neurons accumulating 3H-serotonin or 3H-GABA.     

Modifications of the pyroglutamic acid and histidine residues in thyrotropin-releasing hormone (TRH) yield analogs with selectivity for TRH receptor type 2 over type 1

Thyrotropin-releasing hormone (TRH) analogs in which the N-1(tau) or the C-2 position of the imidazole ring of the histidine residue is substituted with various alkyl groups and the l-pyroglutamic acid (pGlu) is replaced with the l-pyro-2-aminoadipic acid (pAad) or (R)- and (S)-3-oxocyclopentane-1-carboxylic acid (Ocp) were synthesized and studied as agonists for TRH receptor subtype 1 (TRH-R1) and subtype 2 (TRH-R2). We observed that several analogs were selective agonists of TRH-R2 showing relatively less or no activation of TRH-R1. For example, the most selective agonist of the series 13, in which pGlu is replaced with the pAad and histidine residue is substituted at the N-1 position with an isopropyl group, was found to activate TRH-R2 with a potency (EC(50)=1.9microM) but did not activate TRH-R1 (potency>100 microM); that is, exhibited >51-fold greater selectivity for TRH-R2 versus TRH-R1. Analog 8, in which pGlu is replaced with pAad and histidine is substituted at the N-1(tau) position with a methyl group, exhibited a binding affinity (K(i)=0.0032 microM) to TRH-R1 that is similar to that of [Ntau(1)-Me-His]-TRH and displayed potent activation of TRH-R1 and TRH-R2 (EC(50)=0.0049 and 0.0024 microM, respectively). None of the analogs in which pGlu is replaced with the bioisosteric (R)- and (S)-(Ocp) and the imidazole ring is substituted at the N-1(tau) or C-2 position were found to bind or activate either TRH-R1 or TRH-R2 at the highest test dose of 100 microM.     

Specific, water-soluble polypeptides in identified neurons of Aplysia californica.

Application of an ethylene glycol lysis technique to extract water-soluble, low molecular weight polypeptides in Aplysia neurons, was used in conjunction with microgradient gel electrophoresis and micro-isoelectric focusing, to identify unique polypeptides in specific, identified neurons. The polypeptides found in neurons R15, R3-13, R14, and the bag cells were particularly abundant, consistent with the previously suggested neurosecretory role for these cells. Water extraction of the strongly basic polypeptides (pI 10.7) in R3-13 and R14 required an acidic lysis medium.     

Identification and characterization of a novel cell-penetrating peptide

Cell-penetrating peptides (CPPs) are short amino acid sequences that promote their own translocation across cell plasma membrane. When linked with cargo such as polypeptides, nucleic acid, or liposomes, CPPs can facilitate the transport of these entities across the cell membrane. Therefore, CPPs are receiving increased interest in drug delivery and gene therapy. The majority of CPPs identified so far are polycationic peptides which interact with heparin sulfate chains of plasma membrane for internalization. Here, we report the identification and characterization of a conformationally constrained 13 amino acid peptide (CVQWSLLRGYQPC, designated as S41) which is clearly distinct from classical polycationic peptides. Immunofluorescence assay was employed to test the cellular uptake of S41 in mouse neuroblastoma cell line Neuro2A (N2A) and rat cerebellar granule neurons (CGNs). Internalization of S41 was further examined in N2A cells by means of mutational analysis, flow cytometry and confocal microscopy. Our results demonstrate that S41 can enter cells through lipid rafts dependent endocytosis.     

Isolation and partial characterization of neuropeptides that mimic prolonged inhibition produced by bag cell neurons in Aplysia

The bag cell neurons of the marine mollusk Aplysia are part of a neural system that utilizes four neuropeptides as neurotansmitters. The peptides, derived from the egglaying hormone/bag cell peptide (ELH/BCP) precursor protein, are released during a 20-min burst discharge of the bag cells and produce several types of responses in various abdominal ganglion neurons. In the identified neurons L3 and L6, bag cell activity produces prolonged inhibition that lasts for more than 2 h. One of the bag cell peptides, alpha-BCP, mediates an early component of the inhibition in these neurons. To identify the co-transmitter mediating the prolonged component of inhibition, we purified material from an acid extract of abdominal ganglia using molecular sizing high-pressure liquid chromatography (HPLC) on TSK 250-125 followed by two steps of reverse-phase HPLC on C4 or C18. We isolated three inhibitory factors that mimic the prolonged component of inhibition. Mass spectroscopy and partial amino acid sequence analysis indicate one factor is ELH [2-36], that is, ELH that lacks the first, N-terminal amino acid. This inhibitory activity was similar in potency to that of ELH and is the first to be described for an ELH related peptide. The two other factors were approximately 3,300 and 4,700 Da and were effective at 10- and 50-fold lower concentration, respectively, than ELH or its fragment. Amino acid composition analysis suggests that they are not derived from the ELH/BCP precursor protein. The 4,700 Da factor is effective at the lowest concentration and produces an effect that lasts as long as 100 min. Therefore, it is the best candidate for the true inhibitory transmitter. Because the inhibited neurons in nervate the kidney, the function of prolonged inhibition may be to regulate kidney function during egg laying.