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Context: Insulin-like peptide 3 (INSL3), a protein hormone produced by Leydig cells, may play a crucial role in testicular descent as male INSL3 knockout mice have bilateral cryptorchidism. Previous studies have measured human fetal INSL3 levels in amniotic fluid only. Objective: To measure INSL3 serum levels and mRNA in fetal umbilical cord blood and fetal testes, respectively. Design: INSL3 concentrations were assayed on 50 μl of serum from male human fetal umbilical cord blood by a non-commercial highly sensitive and specific radioimmunoassay. For secondary confirmation, quantitative real-time PCR was used to measure INSL3 relative mRNA expression in 7 age-matched human fetal testes. Setting: UT Southwestern Medical Center, Dallas, TX and Medical University of South Carolina, Charleston, SC. Patients or other Participants: Twelve human male umbilical cord blood samples and 7 human male testes were obtained from fetuses 14–21 weeks gestation. Male sex was verified by leukocyte genomic DNA SRY PCR. Interventions: None. Main Outcome Measures: Human male fetal INSL3 cord blood serum concentrations and testicular relative mRNA expression. Results: INSL3 serum concentrations during human male gestational weeks 15–20 were 2–4 times higher than published prepubertal male levels and were 5–100 times higher than previous reports of INSL3 concentrations obtained from amniotic fluid. Testicular fetal INSL3 mRNA relative expression was low from weeks 14–16, rose significantly weeks 17 and 18, and returned to low levels at week 21. Conclusions: These findings further support the role of INSL3 in human testicular descent and could prove relevant in uncovering the pathophysiology of cryptorchidism.

We recently reported that plasma insulin-like peptide 3 (INSL3) concentrations increased soon after endogenous and exogenous stimulations of LH in male goats and bulls. However, the effects of LH suppression on INSL3 secretion are unknown in domestic animals. Here, we examined the effects of a long-acting GnRH antagonist (degarelix acetate; 4 mg/kg) on the secretions of plasma INSL3 and testosterone in two phases, an immediate and a long-term phase in male goats (n = 6; aged, 13-16 months). During the immediate phase, blood was taken at 15-minute intervals for 8 hours on Days -5, 0, and 3. The GnRH antagonist was administered after 2-hour sampling of Day 0. Moreover, a daily blood sample was taken from Day 0 to Day 7, followed by twice a week until 9 weeks and finally at week 10. The scrotal circumference was recorded before treatment and continued biweekly until week 10. Concentrations of LH, INSL3, and testosterone in plasma were determined by EIA and the pulsatile nature of secretion analyzed using pulse XP software. The mean concentrations, pulse frequency (per hour), and pulse amplitude (peak-nadir) of plasma LH and testosterone reduced from pretreatment to posttreatment Day 0 and Day 3 (P 0.05). A decline in mean concentrations, pulse frequency, and pulse amplitude of INSL3 was exhibited on posttreatment Day 3 compared with pretreatment (P 0.01). During long-term sampling, a decline (P 0.01) in plasma testosterone and INSL3 concentrations was observed 1 day after treatment and remained lower until 8.5 weeks after treatment, and thereafter returned to pretreatment levels. A reduction in scrotal circumference was recorded 4 weeks after treatment and remained lower until 10 weeks after treatment (P 0.05). In conclusion, the acute regulation of INSL3 by LH was confirmed by reduction of plasma INSL3 levels within 3 days after GnRH antagonist treatment in male goats. Although the onset of suppression of testosterone was more rapid than that of INSL3, the low levels persisted for 8.5 weeks for both hormones, and subsequently the concentrations returned to pretreatment levels. A significant reduction in testicular size was also observed. The quick, long-lasting, and transient suppression of testosterone and INSL3 after a single injection implies a potential application of this antagonist in reversible long-term chemical castration in male goats.

Recently, it was reported that in bulls secretion of insulin-like peptide 3 (INSL3) in blood occurred in a pulsatile manner and was acutely regulated by LH. In the present study, the acute regulation of plasma INSL3 and its temporal relationships with LH and testosterone were examined in six sexually matured male goats using the following experimental design. (1) After stimulating LH release by administering a GnRH analogue, blood levels of LH, INSL3, and testosterone were monitored at 15-minute intervals for 2 hours followed by hourly intervals up to 8 hours. (2) After activation of the LH receptor by hCG blood levels of INSL3 and testosterone were determine at 15-minute intervals for 2 hours, followed by hourly intervals up to 8 hours, daily intervals up to Day 8, and finally on Day 12. (3) The release of LH, INSL3, and testosterone in normal physiology was established at 15-minute intervals for an 8-hour session. Concentrations of LH, INSL3, and testosterone in plasma were measured by enzyme-immunoassays. After GnRH treatment, mean plasma concentrations of all three hormones increased (P

Recent estimates suggest that >300 million people are afflicted by serious fungal infections worldwide. Current antifungal drugs are static and toxic and/or have a narrow spectrum of activity. Thus, there is an urgent need for the development of new antifungal drugs. The fungal sphingolipid glucosylceramide (GlcCer) is critical in promoting virulence of a variety of human-pathogenic fungi. In this study, we screened a synthetic drug library for compounds that target the synthesis of fungal, but not mammalian, GlcCer and found two compounds [N′-(3-bromo-4-hydroxybenzylidene)-2-methylbenzohydrazide (BHBM) and its derivative, 3-bromo-N′-(3-bromo-4-hydroxybenzylidene) benzohydrazide (D0)] that were highly effective in vitro and in vivo against several pathogenic fungi. BHBM and D0 were well tolerated in animals and are highly synergistic or additive to current antifungals. BHBM and D0 significantly affected fungal cell morphology and resulted in the accumulation of intracellular vesicles. Deep-sequencing analysis of drug-resistant mutants revealed that four protein products, encoded by genes APL5, COS111, MKK1, and STE2, which are involved in vesicular transport and cell cycle progression, are targeted by BHBM., IMPORTANCE Fungal infections are a significant cause of morbidity and mortality worldwide. Current antifungal drugs suffer from various drawbacks, including toxicity, drug resistance, and narrow spectrum of activity. In this study, we have demonstrated that pharmaceutical inhibition of fungal glucosylceramide presents a new opportunity to treat cryptococcosis and various other fungal infections. In addition to being effective against pathogenic fungi, the compounds discovered in this study were well tolerated by animals and additive to current antifungals. These findings suggest that these drugs might pave the way for the development of a new class of antifungals.

application/pdf, Article, Theriogenology. 2015, 84 (9), p.1530-1535

Insulin-like peptide 3 (INSL3) has been used as a testis-specific biomarker for puberty in several species, but the secretory profile of INSL3 during pubertal development in small ruminants is unknown. Here we sought to determine the age-related changes in the plasma concentrations of INSL3 and testosterone and their association with scrotal circumference during pubertal development in five male Shiba goats. Blood samples and scrotal circumference measurement were taken every 2 weeks from week 10 to week 52 of each goat's lifespan. Based on the changes in scrotal circumference, data were grouped into early pubertal (10-22 weeks), late pubertal (22-34 weeks) and post-pubertal (34-52 weeks) categories. The plasma concentrations of testosterone and luteinizing hormone (LH) were measured by enzyme-immunoassays (EIAs), and we used a time-resolved fluorescence immunoassay (TRFIA) to measure plasma INSL3. The biweekly sampling showed that the plasma INSL3 secretions maintained a moderate increase during and after puberty, whereas the plasma testosterone secretions fluctuated over the same period. The comparison of the three age categories revealed a significant increase (p 0.01) in the mean plasma INSL3 concentrations during the late and post-pubertal periods compared to the early pubertal period. There was no difference in the mean plasma testosterone concentrations between the early and late pubertal periods, but a significant increase (p 0.01) was observed during the post-pubertal period compared to early and late pubertal periods. The mean plasma LH concentrations increased significantly (p 0.05) from the early pubertal to late pubertal and from the late pubertal to post-pubertal periods. A significant increase (p 0.05) in the mean scrotal circumference from the early pubertal to late pubertal and from the late pubertal to post-pubertal periods was observed. The R

Amino acid 4-sulfobenzyl esters were employed for the synthesis of peptides in solution. They significantly increase the hydrophilicity of protected intermediates as shown by analytical reversed-phase high performance liquid chromatography and counter-current distribution. General methods for working up are described which permit simple and standardized isolations of products. The use of several coupling methods was demonstrated in the synthesis of Z-Val-Gly-OBzl-SO3NH4. Ion-exchange chromatography was introduced as a selective purification procedure for protected peptide 4-sulfobenzyl esters. A step-wise synthesis of [Leu5]-enkephalin was performed, starting from leucine 4-sulfobenzyl ester. Removal of N-terminal protecting groups produced zwitterionic peptide 4-sulfobenzyl esters. These were readily purified by crystallization from slightly acidic media; no further purification was necessary. The biologically fully active pentapeptide Tyr-Gly-Gly-Phe-Leu was obtained in an overall yield of 30.2%.

Biotinylated porcine relaxins were synthesized and purified to homogeneity. Native porcine relaxin was reacted with biotinyl-epsilon-aminohexanoic acid-N-hydroxysuccinimide ester and the resulting mixture was separated by either ion exchange chromatography on CM-cellulose at pH 5.5 or reversed-phase high performance liquid chromatography. All four possible monosubstituted relaxin derivatives, with substitutions in N alpha A1, N epsilon A7, N epsilon A16, and N epsilon B8, were obtained. All derivatives were fully biologically active and comparative circular dichroism spectroscopy showed no significant differences in their secondary structure.

The preparation of the 4-sulfobenzyl esters of 18 amino acid derivatives is described. This carboxyl protecting group was introduced according to Hubbuch et al. (1980). The caesium or dicyclohexylammonium salts of N-terminal protected amino acids were reacted with 4-(bromomethyl)benzenesulfonate (1). After N-terminal deblocking, the amino acid-4-sulfobenzyl esters were isolated as zwitterions. The protecting group was removable by catalytic hydrogenation and by saponification. The 4-sulfobenzyl esters could be easily converted to amides and hydrazides. They were stable to 2 M hydrogen bromide in acetic acid as well as to a 10-fold excess of trifluoromethane sulfonic acid in trifluoro-acetic acid. The behaviours of +H2-Gly-Phe-Leu-OBzl-SO-3 and the corresponding methyl, benzyl and 4-nitrobenzyl esters were compared under various conditions.

Analogous to insulin, the relaxin-like factor (RLF) must undergo a structural transition to the active form prior to receptor binding. Thus, the C-terminus of the B chain of RLF folds toward the surface of the central B chain helix, causing partial obliteration of the two essential RLF receptor-binding site residues, valine B19 and tryptophan B27. Via comparison of the solution structure of a fully active C-terminally cross-linked RLF analogue with the native synthetic human RLF (hRLF), it became clear that the cross-linked analogue largely retains the essential folding of the native protein. Both proteins exist in a major and minor conformation, as revealed by multiple resonances from tryptophan B27 and adjacent residues on the B chain helix. Notably, the minor conformation is significantly more highly populated in the chemically cross-linked RLF than it is in the hRLF. In addition, compared to the unmodified molecule, subtle differences are observed within the B chain helix whereby the cross-linked derivative shows a reduced level of hydrogen bonding and significant peak broadening at the binding site residue ValB19. On the basis of these observations, we suggest that the solution structure of the native hormone represents an inactive conformer and that a dynamic equilibrium exists between the C-terminally unfolded binding conformation and the inactive conformation of the RLF.

Objective—To determine whether abnormal laxity of hip joints of canine pups with genetic predisposition to hip dysplasia (HD+) is related to ingestion of milk-borne hormones. Animals—7 female Labrador Retrievers with HD+ and 8 with low predisposition to hip dysplasia (HD–) and their offspring. Procedures—Immunoactive relaxin, estrogen, and estrogen precursor concentrations in milk of HD+ lactating bitches and in serum of their pups were compared with those of HD– bitches and pups. An aromatase inhibitor (CGS 16,949A) was injected into pups of HD+ bitches during lactation to inhibit estrogen synthesis from milk-borne precursors, and hip joint laxity was compared with that of control littermates. Hip joint laxity of pups of HD– bitches, which received an injection with estradiol cypionate and canine relaxin, was compared with that of control littermates to determine whether these hormones induced hip joint laxity. Results—High concentrations of estrogens and relaxin were found in milk of HD+ and HD– bitches throughout lactation. Serum concentrations of milk-derived relaxin and total estrogens were similar in all pups, but estradiol-17B was detected only in pups of HD+ bitches. Hip joint laxity was reduced in pups that received CGS 16,949A. Hip joint laxity was increased in pups of HD– bitches that received estradiol cypionate and relaxin. Conclusions and Clinical Relevance—Milk-borne maternal hormones and precursors were absorbed into the circulation of canine neonates and may play a role in hip joint laxity in HD+ pups. Phenotypic expression of hip dysplasia may therefore be preventable by antihormone treatment.

Penaeidins are a diverse family of two-domain antimicrobial peptides expressed in shrimp. Variation in penaeidin sequence results in functional diversity, which was discovered using synthetic reproductions of native penaeidins. An isoform of penaeidin class 3 from Litopenaeus setiferus (Litset Pen3-4) was synthesized using native ligation and compared directly with the synthetic penaeidin class 4 known to be expressed in the same organism. New antimicrobial activity data are included in this review that emphasize differences in effectiveness that are apparent from a direct comparison of two classes. A novel approach to intact penaeidin analysis is presented in the form of Fourier Transform Ion-Cyclotron Resonance Mass Spectrometry, which has implications for the identification of individual penaeidin isoforms without chemical modification or enzymatic cleavage. The new information included in this review helps gather the perspective on relevance of penaeidin diversity to antimicrobial function, the use of synthetic peptides as tools to evaluate specific immune functions and the application of high mass resolution, top-down sequencing methods to the intact analysis of individual penaeidin isoforms.

Recent estimates suggest that >300 million people are afflicted by serious fungal infections worldwide. Current antifungal drugs are static and toxic and/or have a narrow spectrum of activity. Thus, there is an urgent need for the development of new antifungal drugs. The fungal sphingolipid glucosylceramide (GlcCer) is critical in promoting virulence of a variety of human-pathogenic fungi. In this study, we screened a synthetic drug library for compounds that target the synthesis of fungal, but not mammalian, GlcCer and found two compounds [ N ′-(3-bromo-4-hydroxybenzylidene)-2-methylbenzohydrazide (BHBM) and its derivative, 3-bromo- N ′-(3-bromo-4-hydroxybenzylidene) benzohydrazide (D0)] that were highly effective in vitro and in vivo against several pathogenic fungi. BHBM and D0 were well tolerated in animals and are highly synergistic or additive to current antifungals. BHBM and D0 significantly affected fungal cell morphology and resulted in the accumulation of intracellular vesicles. Deep-sequencing analysis of drug-resistant mutants revealed that four protein products, encoded by genes APL5 , COS111 , MKK1 , and STE2 , which are involved in vesicular transport and cell cycle progression, are targeted by BHBM. IMPORTANCE Fungal infections are a significant cause of morbidity and mortality worldwide. Current antifungal drugs suffer from various drawbacks, including toxicity, drug resistance, and narrow spectrum of activity. In this study, we have demonstrated that pharmaceutical inhibition of fungal glucosylceramide presents a new opportunity to treat cryptococcosis and various other fungal infections. In addition to being effective against pathogenic fungi, the compounds discovered in this study were well tolerated by animals and additive to current antifungals. These findings suggest that these drugs might pave the way for the development of a new class of antifungals.

Penaeidins are antimicrobial peptides from shrimp that are constituted by divergent classes of peptide isoforms in an individual organism. Penaeidin sequence variation suggests functional diversity in the host and promises differential activities if applied to treat infections in humans. We have synthesized isoform 4 of penaeidin class 3 from the Atlantic shrimp, Litopenaeus setiferus, by native ligation using three peptide segments. Our synthesis approach led to the discovery of an irreversible side reaction that was successfully suppressed, a discovery, which has particular relevance to the synthesis of cysteine-rich peptides. The antimicrobial activity of full-length penaeidin and the N-terminal proline-rich domain of this isoform were compared with the corresponding peptides of penaeidin class 4 isoform 1 using a wide range of bacteria and fungi. New aspects of penaeidin function are reported that include activity against fungi of the phylum Basidiomycota (Cryptococcus strains), activity against fungi that are pathogenic to humans and effectiveness in the context of antibiotic resistance mechanisms (Cryptococcus and Candida spp.). The proline-rich domain of penaeidin class 4 shows the highest relative antimicrobial activity, while exhibiting no cytotoxicity to human monocytes, and therefore stands out as a potential peptide therapeutic.

Early placental insulin-like protein (INSL4 or EPIL) is a member of the insulin superfamily of hormones, which is highly expressed in the placenta. We have confirmed this at term and shown it to be expressed by the maternal decidua. Although an abundance of locally acting growth factors are produced within the uterus during pregnancy, we hypothesized that INSL4 plays an important role in fetal and placental growth. We have demonstrated with cell lines and primary cells that it has a growth-inhibitory effect by causing apoptosis and loss of cell viability. We used primary amniotic epithelial cells for flow cytometry to show that INSL4 caused apoptosis, which was dose-related and significant (P < 0.05) at 50 ng/ml. This was confirmed by measurement of the nuclear matrix protein in the media. In comparison, relaxin treatment (up to 200 ng/ml) had no effect on apoptosis. The addition of INSL4 (3-30 ng/ml) also caused a loss of cell viability, although it had no effect on the numbers of cells at different phases of the cell cycle. Placental apoptosis is an important process in both normal placental development and in fetal growth restriction. Therefore, an in vivo clinical correlate was sought in fraternal twins exhibiting discordant growth. Expression of the INSL4 gene was doubled in the placenta of the growth-restricted twin compared to the normally grown sibling, suggesting that it may be linked to a higher level of apoptosis and loss of cell viability and, therefore, that it may contribute to fetal growth restriction.

The pleated sheet region of the leucine-rich G-protein-coupled receptor 7 supports a relaxin-binding group of amino acids that perfectly matches the binding cassette of relaxin. Arginines B13 and B17 are each chelated by an aspartic acid/glutamic acid pair and by isoleucine B20, which, offset by a one-quarter helix turn from a straight line connecting the arginines, interacts with a cluster of hydrophobic amino acids. The binding cassette of relaxin cuts at an angle of approximately 45 degrees across five parallel leucine-rich repeats. The arginine residues 13 and 17, which evolve parallel from the B-chain alpha-helix of relaxin, neutralize the charge repulsion of the juxta-posed acidic groups on the receptor and thereby trigger closure of a hydrogen bonding network around the guanidinium groups. Thus, relaxin is bound by synchronized chelation of two arginines and stabilized by hydrophobic interaction of isoleucine B20 with tryptophan, isoleucine, and leucine in neighboring leucine-rich repeats of the receptor. Deletion of any one of the three features diminishes interaction to the level of nonspecific binding. This model explains the exquisite sensitivity of relaxin binding avidity to minute changes in the disposition of the guanidinium and the size dependence of the hydrophobic binding residue in position B20.

The relaxin-like factor (RLF) is thought to be responsible for the intra-abdominal migration of the testis during mammalian development. Our latest studies of RLF and LGR8 have revealed that the N-terminal region of the A chain is not required for receptor binding but is indispensable for cyclic AMP generation. RLF derivatives with six residues deleted from the N terminus of the A chain are active, whereas further truncation, up to the first A chain cysteine (A-10), yields tightly binding ligands devoid of signaling activity. These derivatives are specific competitive inhibitors (RLFi) of RLF. Although receptor binding is dependent upon B chain residues, the N-terminal region of the A chain is a generic trigger of the trans-membrane signaling activity.

Antimicrobial peptide structure has direct implications for the complexity of functions and mechanisms of action. The penaeidin antimicrobial peptide family from shrimp is divided into multiple class designations based on primary structure. The penaeidin classes are not only characterized by variability in primary sequence but also by variation in target specificity and effectiveness. Whereas class 4 exhibits low isoform diversity within species and is highly conserved between species, the primary sequence of penaeidin class 3 is less conserved between species and exhibits considerable isoform diversity within species. All penaeidins, regardless of class or species, are composed of two dramatically different domains: an unconstrained proline-rich domain and a disulfide bond-stabilized cysteine-rich domain. The proline-rich domain varies in length and is generally less conserved, whereas the spacing and specific residue content of the cysteine-rich domain is more conserved. The structure of the synthetic penaeidin class 4 (PEN4-1) from Litopenaeus setiferus was analyzed using several approaches, including chemical mapping of disulfide bonds, circular dichroism analysis of secondary structural characteristics, and complete characterization of the solution structure of the peptide by proton NMR. L. setiferus PEN4-1 was then compared with the previously characterized structure of penaeidin class 3 from Litopenaeus vannamei. Moreover, the specificity of these antimicrobial peptides was examined through direct comparison of activity against a panel of microbes. The penaeidin classes differ in microbial target specificity, which correlates to variability in specific domain sequence. However, the tertiary structure of the cysteine-rich domain and indeed the overall structure of penaeidins are conserved across classes.

All kinetic indicators suggest that the receptor-binding site of the relaxin-like factor (RLF) is located in the flexible C-terminal region of the B chain and is centered about the tryptophan in position B27. Conformational restraints of varying lengths were used to confine Trp (B27) to a narrow range of positions relative to the C terminus of the A chain. Total synthesis of variants involving residues proximate to Trp (B27) assured us that none had a role in receptor binding. Even arginine B26, next to the important tryptophan, can be replaced without deleterious effects. To fix the distance between the C-terminal end of the A chain and Trp (B27) at predetermined lengths, we synthesized RLF with covalent cross-links between a lysine, which was placed in position B26, and the alpha-carboxyl group at the C terminus of the A chain (A26). The affinity of the cross-linked ligands for the RLF receptor varied as a parabolic function of length whereby the range of 10.0-11.1 A provided the closest approach to the binding conformation. Wild-type transmembrane signaling activity, as determined by cAMP accumulation, was reached only with a glycine cross-linker.

To test the hypothesis that relaxin may play a role in the fetal abnormalities associated with pregnancy in type 1 diabetic women, we previously compared gestational relaxin concentrations in diabetic and clinically normal women using a porcine relaxin radioimmunoassay (RIA): Serum immunoactive relaxin was significantly (P < 0.001) elevated in the diabetic women. To confirm and extend this work in a larger group of subjects, we have now used an enzyme-linked immunosorbent assay (ELISA) specific for human H2 relaxin (the normal human gene product) to determine immunoactive serum relaxin concentrations in serial samples from 61 Type 1 diabetic and 21 normal pregnant women. Samples from 22 of the diabetic and nine of the normal women were also directly compared in the porcine relaxin RIA. ELISA-determined serum relaxin was higher (P < 0.001) at 24 and 36 weeks of pregnancy in type 1 diabetic women than in controls, confirming previous findings. However, the geometric mean increase in immunoactive relaxin concentration in identical samples from pregnant diabetic women over that of controls was significantly greater with the RIA than with the ELISA (271% vs 44%; P < 0.001). To investigate this discrepancy, the specificity and epitope selectivity of the RIA and the ELISA were compared using several synthetic polypeptides, including human relaxins H1 and H2, and relaxin and insulin derivatives. Both assays showed great specificity, but the porcine RIA selectively identified the epitopes of the receptor-binding domain of the relaxin B chain and cross-reacted strongly with H1 and H2 relaxins. In contrast, only the H2 peptide was detected by the ELISA antiserum. Therefore, the marked discrepancy between the RIA and the ELISA could be due to the presence in the diabetic samples of another relaxin-like molecule in addition to the normal H2 relaxin. The biological consequences of elevated serum relaxin in diabetic pregnancy remain to be elucidated.

Testicular descent in mice is dependent upon proper outgrowth of the gubernaculum primordia under the influence of the insulin-like 3 gene product (Insl3). Deletion of this gene prevents gubernaculum growth and causes bilateral cryptorchidism. In vitro experiments have led to the suggestion that Insl3 and androgens together induce outgrowth of the gubernacular primordia. The experiments reported here were designed specifically to determine whether in vivo the Insl3-mediated gubernaculum development is independent of androgens. To that effect transgenic male and female mice were generated that overexpressed Insl3 in the pancreas during fetal and postnatal life. Expression of the transgenic allele in the Insl3-deficient mice rescued the cryptorchidism in male mutant, indicating that the islet beta-cells efficiently processed the Insl3 gene product to the functional hormone. All transgenic females displayed bilateral inguinal hernia. The processus vaginalis developed containing intestinal loops. The Müllerian derivatives gave rise to oviduct, uterus, and upper vagina, and Wolffian duct derivatives were missing, indicating the absence of the androgen- and anti-Müllerian hormone-mediated activities in transgenic females. The ovaries descended into a position over the bladder and attached to the abdominal wall via the well developed cranial suspensory ligament and the gubernaculum. Administration of dihydrotestosterone during prenatal development suppressed formation of the cranial suspensory ligament and thereby allowed the descent of the ovaries into the processus vaginalis. These results suggest that Insl3-mediated activity induces gubernaculum development and precludes a role of androgen in this process. Furthermore, the transgenic females exhibit reduced fertility, which is due to fetal mortality during midgestation.

A series of neurotensin (NT)(8-13) analogs featuring substitution of the Arg8 and/or Arg9 residues with non-natural cationic amino acids was synthesized and evaluated for binding to the human NT receptor-1 (hNTR-1). The modifications were designed to probe specific steric and electrostatic requirements in the N-terminal cationic region of NT(8-13) for receptor binding as a general evaluation of the feasibility of incorporating minor structural changes into a peptide at a crucial polar receptor binding site. Many of the non-natural amino acids are more or less isosteric to Arg but more lipophilic as a result of addition of alkyl groups or through removal or replacement of NH character with methylene or methyl substituents, whereas others vary the distance between the cation and the alpha-amino acid carbon. Substitution of Arg8 with N(G)-alkylated Arg derivatives or homolysine (Hlys) maintained the subnanomolar affinity of NT(8-13) to the hNTR-1. Position 8 incorporation of Hlys produced the most favorable primary amine side-chain substitution to date. Moderate losses in affinity observed with position 9 substitutions were attributed to adverse steric effects. Doubly substituted [Hlys8, DAB9]NT(8-13), in which DAB is 2,4-diaminobutyric acid, was also prepared and tested as the shorter side-chain of DAB is known to be favored in position 9 of NT(8-13). This analog maintained 60% of NT(8-13) binding affinity making it the most favored des-guanidinium-containing analog known. These results demonstrate that adequate receptor binding affinity can be maintained over a structural range of Arg analogs, thus providing a range of peptides expected to exhibit altered pharmacokinetic properties. From the standpoint of the hNTR-1 cationic binding sites, these results help to map out the structural stringency inherent in the formation of a tight binding complex with NT(8-13) and related analogs.

Deletion of the relaxin-like factor (RLF) gene in mice causes retention of testicles and infertility. The development of a synthetic RLF has made it possible to investigate the events that connect the genomic event and the basic biological responses that cause gonadal positioning. Anti-RLF antibodies were raised against synthetic RLF, allowing determination of RLF concentrations during the critical period, testing for RLF receptors on the gubernaculum and exploration of the temporal relationship between receptor display and migration of the testes in developing rats. In male rat pups, serum RLF concentrations were high at day 2 before parturition (2.4 ng ml(-1)) and decreased sharply just before parturition. Thereafter, males and females had the same low serum concentrations until RLF concen-trations began to increase in males only, starting at day 10 after parturition and continuing until adult RLF concentrations (0.6 ng ml(-1)) were reached on day 39 after parturition. The testicles are descending into the scrotum during this phase of increasing RLF concentrations and are descended fully by day 19-21 after parturition, before adult hormone concentrations are established. The high prenatal serum RLF concentration coincides with high expression of RLF receptors in the gubernaculum tissue. Competitive binding of RLF per mg of membrane protein prepared from rat gubernacula at various developmental stages showed no increase in receptor density as sexual maturity was reached. Gubernaculum cells in primary culture showed an increased uptake of 5-bromo-2'-deoxyuridine in the presence of RLF compared with controls. These studies demonstrate that the synthetic RLF is biologically active and indicate that the cryptorchid phenotype INSL3(-/-) is a direct consequence of defective gubernaculum growth, caused by the absence of RLF during early phases of development.

Insulin-like 4 (INSL-4) is a protein expressed in the early placenta. Its primary structure is insulin-like with reference to the distribution of cysteine residues and the single chain pro-form. Insulin-like 4 was generated by solid-phase peptide synthesis of the two chains followed by the sequential synthesis of the three disulfide bonds. Two disulfide isomers were produced, one with an insulin-like disulfide bonding pattern and the other with a reversed chain orientation. The CD spectra of the two disulfide isomers were indistinguishable without any features produced by periodic structures. In addition, the hydrodynamic properties of the two isomers were identical which implied a very open structure of the disulfide-bonded two-chain molecules. It appears that insulin-likeness cannot be defined solely on the basis of the primary structure of cDNA.

Relaxin has a unique, clearly identifiable, mixed function receptor-binding region comprising amino acid residues that evolve sequentially from the central portion of the B chain alpha-helix. Two arginine residues in positions B13 and B17 that project like forefinger and middle finger from the helix provide the electrostatic element opposed by the hydrophobic (thumb) element isoleucine (B20), offset from the arginines by about 40 degrees. The binding intensity of relaxin to its receptor decreases by 3 orders of magnitude if alanine is substituted for the newly discovered binding component isoleucine in position B20. The arginine residues cannot be replaced by other positive charges, nor can the guanidinium group be presented on a longer or shorter hydrocarbon chain. In contrast, the hydrophobic interaction is incremental in nature, and the contribution to the total binding energy is roughly proportional to the number of hydrocarbon units in the side chain. It appears that a hydrophobic surface exists on the receptor that offers optimal van der Waals' interaction with beta-branched hydrophobic amino acids. The binding energy increases roughly 10-fold with each methylene group whereby beta-branching is more effective per surface unit than chain elongation. Aromatic side chains appear to demarcate the extent of the binding region in so far as residues larger than phenylalanine decrease receptor binding. The exceptional clarity of binding site geometry in relaxin makes for an excellent opportunity to design peptido-mimetics.

Bombyxin is an insect hormone with an insulin-like structure which affects the reduction of stored carbohydrates in the silkworm Bombyx mori. The receptor binding surface of bombyxin includes a trough on the interface between the B chain helix and the N-terminal A chain helix. Alanine(B15) is located on the edge of this feature, whereas the bottom is formed by hydrophobic core residues Ile(A2) and Leu(B14). Replacement of alanine(B15) with bulkier residues produces a negative steric effect on bombyxin receptor binding; alpha-aminobutyric acid reduced the affinity to 6.5%, valine to 1.1%, norvaline to 0.88%, and leucine to 0.05%. CD spectra of these analogues were indistinguishable from each other and identical to that of bombyxin. Changing the backbone structure by replacing alanine with glycine and alpha-aminoisobutyric acid resulted in analogues with activities of 3.7 and 1.4%, respectively, but also a disturbed structure as determined by CD spectroscopy. Replacement of other residues on the periphery of the trough, i.e., arginines at positions B12 and B16, also reduced the level of receptor binding but to a lesser extent than the replacement of alanine(B15). The level of receptor binding for citrulline(B12) bombyxin was 17% and for citrulline(B16) bombyxin was 45%. When it is considered that glycine(A1) is located on the edge of the same trough but across from Ala(B15) and is required for maintenance of the overall structure of bombyxin, it is proposed that the bombyxin receptor binding site forms a contiguous hydrophobic area consisting of residues Ile(A2), Leu(B14), and Ala(B15).

The relaxin-like factor (RLF) is a circulating hormone that binds to specific membrane-bound uterine receptors in the mouse. Mono-iodinated RLF tracers were produced and characterized specifically to study the properties of the RLF receptor. The tracers bound to the RLF receptor in uterine crude membrane preparations with high affinity (73 nm for 125I-Tyr(A9) RLF and 90 nm for 125I-Tyr(A26) RLF) as determined by Scatchard analysis. The specificity of binding was confirmed by chemical cross-linking experiments. Binding of 125I-Tyr(A9) RLF to the putative receptor was inhibited in the presence of a 640-fold excess of unlabeled human RLF but not by the same excess of human relaxin. SDS-gel electrophoresis of the RLF-receptor complex revealed a molecular mass of >200 kDa, which remained unchanged upon reduction. The size and the lack of subunit structure of the receptor is similar to the features reported for the relaxin receptor. In this regard both, the RLF and the relaxin receptor are different from the insulin- and the insulin-like growth factor-type 1 receptors. This observation supports the relaxin-likeness of this new factor not only toward potential target tissues but also as regards receptor features.

Bombyxin is an insect neurohormone with an insulin-like structure. The N-terminal A chain helix, a region which is considered part of the active site in insulin, is almost identical between the two hormones. Bombyxin analogues with modifications at the N-terminus of the A-chain were synthesized and investigated for their ability to bind to bombyxin-specific receptors. While N-acetylation reduced the affinity to the bombyxin receptor to 18% the removal of glycine (A1) inactivated the hormone completely. Replacement of glycine (A1) by l-amino acids caused a significant loss in activity (11%) while its replacement by d-amino acid resulted in active bombyxin analogues (55%). Comparative CD spectroscopy indicated a change in structure for desGly(A1)bombyxin. Although the insect hormone does not have an insulin-like function it exhibits mammalian insulin-like structural sensitivity for A chain modifications.

The relaxin-like factor (RLF) is a circulating hormone that is synthesized in the gonads of mammals and released into the bloodstream. The distribution of its receptor and a trace of cross-reactivity to relaxin receptors implied that this relatively new factor is more relaxin- than insulin-like. The chemical synthesis of RLF analogues with specific modifications in positions B27 and B25, or the truncated form des(B27-31)RLF, clearly indicate that the intact indole ring in position B27 is crucial for high RLF receptor-binding. Receptor-binding was reduced by 2 orders of magnitude for Leu(B27)RLF (3%), Ala(B27)RLF (2.1%), and des(B27-31)RLF (0.4%), whereas slightly better binding was observed for His(B27)RLF (7.5%), Phe(B27)RLF (21%), D-Trp(B27) (26%), and the oxindole(B27)RLF (41%). On the basis of these observation it seems that an aromatic ring system in the beta- or gamma-position is required for binding. Structure prediction of the C-terminal region of the B chain indicated a possible type I or type III turn for residues C-G-G-P-R (B22-26) preceding the tryptophan. Exchanging Pro(B25) for D-Pro within the turn caused a severe structural rearrangement at the C terminus and a 96% drop in activity. It appears that the steric effect of L-Pro(B25) is important for the proper positioning of Trp(B27).

A series of protected ω-mercapto amino acids with side-chain lengths ranging from 3–5 methylene units has been synthesized via nucleophilic substitution of ω-bromo-α-azido acids by 4-methoxy-α-toluenethiol followed by reduction of the azido functionality with SnCl 2 . These enantiomerically pure protected cysteine analogues can be used to optimize the length of disulfide connections in cyclically constrained peptide pharmacophores.

The alkaline gland fluid of the Atlantic stingray (Dasyatis sabina) contains a molecule that cross-reacts weakly to anti-porcine relaxin antibodies. This material was isolated and purified to homogeneity by reversed-phase high-performance liquid chromatography. In SDS gel electrophoresis, the molecule showed an apparent molecular mass of 13 kDa which upon reduction formed two polypeptide chains of 4 and 9 kDa, respectively. Sequence analyses revealed a 27-amino acid residue A chain and a 54-amino acid residue B chain which contained an N-glycosylation site in position B37. The distribution of the six cysteines and possibly the disulfide bonding is identical to that found in insulins and most relaxins. Although the stingray relaxin-like molecule contains the structurally relevant glycine residues within the A chain, in the midregion of the B chain it has only one of the two requisite binding site arginines, which explains the lack of relaxin bioactivity in standard mammalian assays. Stingray relaxin is the first member of the relaxin family identified in a nonhomeotherm male. Carbohydrate analysis of relaxin revealed an N-linked asialo, agalacto, bisected biantennary, and a core-fucosylated oligosaccharide in the position of Asn B37 which makes it the first reported glycosylated relaxin-like molecule.

Rat relaxin, as isolated from ovaries, has been described in the literature as a low potency hormone in the mouse symphysis pubis assay. Searching for an explanation, a helix-breaking glycine residue in the B chain seemed to be the most auspicious perturbation. Rat relaxin was chemically synthesized and analyzed by reverse-phase high performance liquid chromatography, amino acid composition, mass spectrometry and circular dichroic spectroscopy. Analogs of rat relaxin were synthesized either with aspartic acid in place of the helix-breaking glycine residue in the receptor-binding region of the B chain or with Asp-Leu-Val instead of Gly-Tyr-Val at positions B14-B16. In receptor-binding assays [B14D, B15L, B16V]relaxin was a better ligand than rat relaxin, whereas the [B14D]relaxin was less potent. In the mouse symphysis pubis assay, both analogs were less potent than unmodified rat relaxin, but the [B14D, B15L, B16V]relaxin was better than [B14D]relaxin. In contrast to previous reports on native rat relaxin, the chemically synthesized rat relaxin proved to be as active as human and porcine relaxin with respect to the standard mouse assay system. Glycine, which is considered to be a perturbator in an alpha helix, is not only tolerated in the B14 position but is required for full biological potency.

Canine relaxin (cRlx) was synthesized by a combination of solid-phase methods and sequential site-directed disulfide bond formation. Proof that the intended molecule had been synthesized was obtained by analytical HPLC of the intact and reduced molecule, by amino acid and sequence analysis, and by receptor binding and in vivo mouse interpubic ligament bioassays. Antisera to synthetic cRlx were raised in six male rabbits; these cross-reacted with relaxins of other species, but not with insulin, LH, FSH, hCG, or prolactin (PRL). Three of the antisera neutralized relaxin-induced interpubic ligament formation in estrogen-primed mice in vivo. A new homologous cRlx RIA was developed through the use of rabbit antiserum 79888, synthetic cRlx for standards and 125l-labeled trace, and a goat anti-rabbit lgG-polyethylene glycol precipitant. The new RIA can be completed in 26 h and has a sensitivity of 0.195-0.39 ng cRlx/tube. Intra- and interassay coefficients of variation were 3% and 12.5%. During pregnancy in bitches, serum cRlx rose to about 10 micrograms/ml. Immunoactive cRlx was also detected in serum, colostrum, and milk of lactating bitches, but not in large volumes (100-300 microliters) of serum of pseudopregnant or estrous bitches. Immunoreactive cRlx was also found in seminal plasma, but not in serum, of male dogs. The new homologous cRlx RIA is simple, rapid, sensitive, and specific, and will be used in future studies of canine relaxin physiology.

The relaxin-like factor (RLF) also named insulin-like 3 (INSL3) consists of two polypeptide chains linked by two interchain and one intrachain disulfide bond. RLF binds to its receptor (LGR8 also named RXFP2) through the B chain and initiates transmembrane communication by activating the adenylate cyclase through the N-terminal region of both chains. Cystine A11-B10 occupies a unique position on the molecular surface just outside the binding region and between the two signaling ports. We have synthesized an RLF analogue in which the disulfide A11-B10 was replaced by a peptide bond and found that cAMP production ceased while receptor binding was not affected. In contrast, replacing the disulfide A24-B22 by a peptide bond reduced potency proportional to the binding affinity and lowered efficacy to 65%, while replacing disulfide A10-A15 by a peptide bond reduced binding affinity to 32% and lowered potency to 7% but maintained 100% efficacy. The exceptional properties of the derivative bearing an A11-B10 isopeptide cross-link suggests that the disulfide has a special role in signal transduction. We propose that disulfide A11-B10 serves as an insulator between the two ports, whereas the amide functionality disturbs the signal transmission complex likely due to changes in polarity. The clear separation between receptor binding and signal activation sites within this small protein permits one to study how the relaxin-like factor initiates the signal on the receptor that induces intracellular cAMP production.

Testicular Leydig cells secrete insulin-like peptide 3 (INSL3) and express its receptor, RXFP2. However, the effects of INSL3 on endocrine function of Leydig cells are unknown. The present study examines the effects of INSL3 on mouse Leydig cells taking testosterone and cAMP secretions as endpoints. Leydig cells were isolated from testicular interstitial cells obtained from 8-week-old male mice. Cells were then plated in the presence or absence of mouse, human, canine or bovine INSL3 (0-100 ng/ml) for 18 h in multiwell-plates (96 wells) in different cell densities (2500, 5000, 10,000 or 20,000 cells per well). The effects of bovine INSL3 (100 ng/ml) on testosterone secretion by Leydig cells were examined in the presence or absence of, an adenylate cyclase inhibitor, SQ 22536 (1μM) or INSL3 antagonist (bovine and human; 100 ng/ml). Testosterone and cAMP in spent medium were measured by enzyme immunoassay. All INSL3 species tested significantly stimulated the testosterone secretion in Leydig cells, and the maximum stimulation was observed with 100 ng/ml bovine INSL3 at the lowest Leydig cell density (2500 cells per well). Moreover, bovine INSL3 (100 ng/ml) significantly stimulated the cAMP production from Leydig cells maximally at 1h, and remained significantly elevated even at 18 h. SQ 22536 and INSL3 antagonists (bovine and human) significantly reduced INSL3-stimulated testosterone secretion from Leydig cells. Taken together, stimulatory effects of INSL3 on testosterone secretion in Leydig cells are exerted via the activation of cAMP, suggesting a new autocrine function of INSL3 in males.

Relaxin and insulin are disulfide homologues with divergent functions and antigenicity. We have synthesized human relaxin II and porcine insulin and several A chain loop variants of each and measured the effect of substitutions in vivo and in vitro. Substitution of IleA10 in insulin with glycine reduced the receptor binding ability by 2 orders of magnitude. Conversely, exchange of the glycine A14 residue in relaxin, which corresponds to the insulin position A10 for isoleucine, reduced the bio-activity and the receptor binding capacity of relaxin about 100-fold. Substitution of L-Ala in insulin as well as relaxin represented a compromise that allowed both hormones to recover about 30% of the native potency. X-ray analysis and computer-derived energy calculation confirm our receptor binding and biological potency studies, which suggest that the functional difference between derivatives and native hormones is based upon a structural change introduced into the A chain loop by substitution of the penultimate intrachain loop residue. In order to achieve a conformation that favors dynamic or passive interaction with the receptor, insulin and relaxin require a different A chain loop structure in spite of the striking overall similarity.

The objectives were to: (1) develop a time-resolved fluorescence immunoassay (TRFIA) to measure insulin-like peptide 3 (INSL3) in canine plasma; (2) investigate changes of plasma concentrations of INSL3 and testosterone with age in normal male dogs; and (3) compare hormonal concentrations among cryptorchid, normal, and castrated dogs to evaluate endocrine function of the Leydig cell component in retained testes. Blood samples were taken from normal male dogs from prepubertal age to advanced age (4 mo to 14 y, n = 89), and from unilateral cryptorchid (n = 31), bilateral cryptorchid (n = 7), and castrated dogs (n = 3). Canine plasma INSL3 was measured with a newly developed TRFIA. The minimum detection limit of the INSL3 assay was 0.02 ng/ml and the detection range was 0.02 to 20 ng/ml. Plasma INSL3 concentrations increased (P0.05) from prepubertal age (4-6 mo) to pubertal age (6-12 mo), and then declined (P0.05) from pubertal age to post-pubertal age (1-5 y), reaching a plateau. Plasma testosterone concentrations increased (P0.0001) dramatically from prepubertal to pubertal ages, and then seemed to plateau. Concentrations of both INSL3 and testosterone were lower (P0.0001 for each) in bilateral cryptorchid dogs than in normal and unilateral cryptorchid dogs. The INSL3 (range: 0.05-0.43 ng/ml) and testosterone (range: 0.10-0.94 ng/ml) concentrations were readily detected in bilateral cryptorchids, but not in castrated dogs (INSL30.02 ng/ml; testosterone0.04 ng/ml). In conclusion, plasma INSL3 concentrations in male dogs measured by a newly developed TRFIA had a transient surge at a pubertal age, whereas testosterone did not. Lower plasma concentrations of INSL3 and testosterone in bilateral cryptorchid dogs suggest impaired endocrine functions of Leydig cell component in paired retained testes. Therefore, peripheral plasma INSL3 and testosterone concentrations have potential diagnostic value in predicting the presence of bilaterally retained testes in male dogs.

The objectives were to: (1) develop an enzyme immunoassay (EIA) for insulin-like peptide 3 (INSL3) or relaxin-like factor (RLF) in bovine plasma; (2) investigate changes of plasma INSL3 concentrations from birth to pubertal age of beef bulls; and (3) compare changes in plasma concentrations of INSL3, testosterone, and LH. Plasma samples were collected from beef bull calves (n = 15) at birth (0 d) and at 28, 56, and 84 d after birth. Furthermore, in beef bulls around pubertal age (n = 26; age range 3 to 22 mo), plasma samples were collected at 1 to 4 mo intervals. Plasma INSL3 concentrations increased (P < 0.05) from 0 to 28, 28 to 56, and from 56 to 84 d of age. Plasma testosterone concentrations increased (P < 0.001) from 0 to 28 d, and from 28 to 56 d, but did not change from 56 to 84 d. For bulls around pubertal age, plasma INSL3 concentrations did not change from the prepubertal phase (3 to 6 mo) to the early pubertal phase (6 to 12 mo), but increased (P < 0.05) from the early to late pubertal phases (12 to 18 mo), and from the late pubertal to postpubertal phases (18 to 22 mo). Plasma testosterone concentrations increased from the prepubertal to early pubertal phases (P < 0.001), but did not change thereafter. Plasma LH concentrations did not change from 0 d to 84 d, but decreased (P < 0.001) from prepubertal to early pubertal phase, with no significant change thereafter. Plasma INSL3 concentrations increased during the first 3 mo of life and throughout the pubertal age in beef bulls. There were similar dynamic patterns for INSL3 and testosterone during the first 3 mo of life, but patterns subsequently diverged in bulls around pubertal ages.

The chemically synthesized DNA-coding sequence for an artificial single chain human relaxin consisting of a B chain, an Arg-Arg-Glu-Phe-Lys-Arg-connecting peptide, followed by the A chain, was used to construct two plasmids which were introduced into Saccharomyces cerevisiae . Expression of the relaxin-coding sequence was under the control of either the yeast TDH3 promoter or the CUP1 promoter. The yeast α-factor signal sequence was used to direct the protein into the secretory path, and the appearance of human relaxin in the growth medium was confirmed by radioimmunoassay and immunoblotting. Partially purified human relaxin from yeast was biologically active in the mouse symphysis pubis assay and radioreceptor assay.

LH receptor knockout (LhrKO) male mice exhibit a bilateral cryptorchidism resulting from a developmental defect in the gubernaculum during the inguinoscrotal phase of testis descent, which is corrected by testosterone replacement therapy (TRT).In vivoandin vitroexperiments were conducted to investigate the roles of the androgen receptor (AR) and RXFP2 signals in regulation of gubernacular development inLhrKO animals. This study demonstrated that AR and RXFP2 proteins were expressed in the gubernaculum during the entire postnatal period. TRT normalized gubernacular RXFP2 protein levels inLhrKO mice. Organ and primary cell cultures of gubernacula showed that 5α-dihydrotestosterone (DHT) upregulated the expression ofRxfp2which was abolished by the addition of an AR antagonist, flutamide. A single s.c. testosterone injection also led to a significant increase inRxfp2mRNA levels in a time-dependent fashion inLhrKO animals. DHT, natural and synthetic insulin-like peptide 3 (INSL3), or relaxin alone did not affect proliferation of gubernacular mesenchymal cells, while co-treatments of DHT with either INSL3 or relaxin resulted in an increase in cell proliferation, and they also enhanced the mesenchymal cell differentiation toward the myogenic pathway, which included a decrease in a mesenchymal cell marker, CD44 and the expression of troponin. These effects were attenuated by the addition of flutamide, siRNA-mediatedRxfp2knockdown, or by an INSL3 antagonist. Co-administration of an INSL3 antagonist curtailed TRT-induced inguinoscrotal testis descent inLhrKO mice. Our findings indicate that the RXFP2 signaling pathway plays an important role in mediating androgen action to stimulate gubernaculum development during inguinoscrotal testis descent.

Recent structure/function studies on human relaxin II have led to the conclusion that the arginines B13 and/or B17 are important for biological activity. These studies have been confirmed and extended with the help of chemically synthesized derivatives, i.e. dicitrulline (B13, B17), two monocitrulline (B13 and B17), a dilysine (B13, 17), and alanine (B17) relaxins. The CD spectra of synthetic human relaxin and of the derivatives are indistinguishable. Yet, only the native human relaxin II is biologically active and binds strongly to relaxin receptor preparations in vitro. The inactivation is strictly due to side chain functions, in particular the replacement of either or both arginines in the positions B13 or B17. Binding is mediated by a two-prong electrostatic and hydrogen-binding interaction via arginines B13 and B17. Neither B13 nor B17 alone are sufficient and a positive charge equidistant from the B chain helix is equally insufficient. This binding mechanism appears to be unique, as concerns hormone receptor interaction.

Bovine rhodopsin has been reported to be S-palmitylated at cysteines 322 and 323 (Ovchinnikov, Y. A., Abdulaev, N. G., and Bogachuk, A.S. (1988) FEBS Lett. 230, 1-5). Using a combination of enzymatic and chemical cleavage techniques in conjunction with tandem mass spectrometry, the sites of incorporation of the palmityl groups are shown. Bovine rhodopsin in disc membranes was digested with thermolysin to generate the C-terminal fragment (241-327), which was subsequently cleaved with cyanogen bromide to generate the peptide Val-Thr-Thr-Leu-Cys-Cys-Gly-Lys-Asn-Pro (318-327). A bis-S-palmitylated synthetic standard had the same retention time by reversed-phase high performance liquid chromatography as the isolated peptide and the same molecular weight (MH+1511.7) by liquid secondary ion mass spectrometry. Dithiothreitol reduction of both the isolated and the synthetic peptide cleaved the two thioester-linked palmityl groups to produce reduction products of the same appropriately decreased molecular weight (MH+1035.5). Tandem mass spectrometry of the isolated and the synthetic peptide identified the sites of attachment of the palmityl groups on cysteines 322 and 323. These results prove the modification of cysteines 322 and 323 with palmitic acid in bovine rhodopsin, and illustrate the utility of mass spectrometry to characterize the post-translational modifications in G-protein coupled receptors.

The synthesis of an unsymmetrical cystine-derivative (Nα′-9-fluorenyl-methyloxycarbonyl-Nα″-tert.butyloxycarbonyl-cystine-O′-benzyl ester) allowed the sequential synthesis of a disulfide-linked two-chain polypeptide on solid-phase.

The discovery of the total separation of receptor-binding and signal-generating regions of the relaxin-like factor (RLF) has provided an opportunity to investigate the mechanism of transmembrane signaling. The receptor-binding residues of RLF are in the B chain of the two-chain molecule and extend from the midregion of the central helix to the tryptophan in position B27. The signal initiation site resides in two residues before the N-terminal cysteine in each chain. For optimal signaling the RLF requires five L-alpha-amino acids preceding cysteine A10, whereas the B chain requires only three. The nature of the side chains of these amino acids is not critical for the signaling function. Heuristic arguments lead us to suggest that the peptide bond is the signal-generating feature of RLF and possibly of other peptide hormones.

Plasma desorption mass spectrometry (PDMS) was used to determine the C-terminus of the B-chain porpoise relaxin, a polypeptide hormone having three dimensional and disulfide homology with insulin. Trypsin was used to generate a series of peptide fragments for mapping by PDMS. The C-terminal fragments were purified and their sequences determined by on-foil digestion with carboxypeptidase Y.

Human relaxin, a two-chain protein hormone, was synthesized by solid-phase peptide synthesis in combination with a novel thiol-protecting group strategy whereby the three disulfide bonds could be synthesized sequentially and without error. The final product was shown to be homogeneous by reversed-phase high performance liquid chromatography and electrophoresis and had the correct amino acid composition and sequence. Tryptic digestion and peptide mapping of the synthetic relaxin by reversed-phase high performance liquid chromatography resulted in a pattern identical with that produced by standard tryptic relaxin fragments synthetized by different methods. Three human relaxin derivatives containing oxidized methionine, formyltryptophan, and bis[B13,B17-citrulline]-relaxin, were produced and their biological activity and structural similarity to human relaxin was assessed. All derivatives, except those containing modified tryptophan residues, showed indistinguishable circular dichroic spectra, indicating that the modifications did not cause significant structural changes. However, only human relaxin and the tryptophan- and methionine-protected relaxin derivatives showed bioactivity. The derivative in which the two arginines in positions B13 and B17 had been replaced by the uncharged isosteric amino acid citrulline were biologically inactive. This observation confirms preliminary studies (Bullesbach, E. E. and Schwabe, C. (1988) Int. J. Pept. Protein Res. 32, 361-367) that suggested that these two conserved arginines located in the midregion of the relaxin B chain are essential for the function of the hormone.