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Profound neuropeptide diversity characterizes the nematode nervous system, but it has proven challenging to match neuropeptide G protein-coupled receptors (GPCR) with their cognate ligands in heterologous systems. We have expressed the Caenorhabditis elegans GPCR encoded in the locus T19F4.1, previously matched with FMRFamide-like peptides encoded on the flp-2 precursor gene, in mammalian cells and in the yeast Saccharomyces cerevisiae. Pharmacological characterization revealed that the receptor is potently activated by flp-2 peptides in CHO cells (∼10 nM EC50) and in yeast (∼100 nM EC50), signaling through a Gqα pathway in each system. The yeast GPCR expression system provides a robust assay for screening for agonists of the flp-2 receptor and is the target of an ongoing high-throughput screening exercise.

Two alternatively spliced variants of an orphan Caenorhabditis elegans G-protein-coupled receptors (GPCRs; Y58G8A.4a and Y58G8A.4b) were cloned and functionally expressed in Chinese hamster ovary (CHO) cells. The Y58G8A.4a and Y58G8A.4b proteins (397 and 433 amino acid residues, respectively) differ both in amino acid sequence and length of the C-terminal tail of the receptor. A calcium mobilization assay was used as a read-out for receptor function. Both receptors were activated, with nanomolar potencies, by putative peptides encoded by the flp-18 precursor gene, leading to their designation as FLP-18R1a (Y58G8A.4a) and FLP-18R1b (Y58G8A.4b). Three Ascaris suum neuropeptides AF3, AF4, and AF20 all sharing the same FLP-18 C-terminal signature, -PGVLRF-NH(2), were also potent agonists. In contrast to other previously reported C. elegans GPCRs expressed in mammalian cells, both FLP-18R1 variants were fully functional at 37 degrees C. However, a 37 to 28 degrees C temperature shift improved their activity, an effect that was more pronounced for FLP-18R1a. Despite differences in the C-terminus, the region implicated in distinct G-protein recognition for many other GPCRs, the same signaling pathways were observed for both Y58G8A.4 isoforms expressed in CHO cells. Gq protein coupling seems to be the main but not the exclusive signaling pathway, because pretreatment of cells with U-73122, a phospholipase inhibitor, attenuated but did not completely abolish the Ca(2+) signal. A weak Gs-mediated receptor activation was also detected as reflected in an agonist-triggered concentration-dependent cAMP increase. The matching of the FLP-18 peptides with their receptor(s) allows for the evaluation of the pharmacology of this system in the worm in vivo., ((c) 2007 Wiley Periodicals, Inc.)

We report the characterisation of the first neuropeptide receptor from the phylum Platyhelminthes, an early-diverging phylum which includes a number of important human and veterinary parasites. The G protein-coupled receptor (GPCR) was identified from the model flatworm Girardia tigrina (Tricladida: Dugesiidae) based on the presence of motifs widely conserved amongst GPCRs. In two different assays utilising heterologous expression in Chinese hamster ovary cells, the Girardia GPCR was most potently activated by neuropeptides from the FMRFamide-like peptide class. The most potent platyhelminth neuropeptide in both assays was GYIRFamide, a FMRFamide-like peptide known to be present in G. tigrina. There was no activation by neuropeptide Fs, another class of flatworm neuropeptides. Also active were FMRFamide-like peptides derived from other phyla but not known to be present in any platyhelminth. Most potent among these were nematode neuropeptides encoded by the Caenorhabditis elegans flp-1 gene which share a PNFLRFamide carboxy terminal motif. The ability of nematode peptides to stimulate a platyhelminth receptor demonstrates a degree of structural conservation between FMRFamide-like peptide receptors from these two distinct, distant phyla which contain parasitic worms.

Despite a simple nervous system, the free-living nematode Caenorhabditis elegans exhibits complex behaviors. The identification of peptide ligands for a G-protein-coupled receptor (GPCR) has provided insight into the neuronal circuitry involved in the regulation of feeding behavior in this worm. Progress in this regard has been accelerated by the discovery that functional expression of worm GPCRs in mammalian cells can be highly temperature dependent. Gene silencing and behavioral analysis has further identified several putative peptide GPCRs that are implicated in reproduction and locomotion. These studies suggest that these peptide GPCRs are legitimate targets for the discovery of novel anthelmintic agents.

This report describes the cloning and functional annotation of a Caenorhabditis elegans orphan G-protein-coupled receptor (GPCR) (C10C6.2) as a receptor for the FMRFamide-related peptides (FaRPs) encoded on the flp15 precursor gene, leading to the receptor designation FLP15-R. A cDNA encoding C10C6.2 was obtained using PCR techniques, confirmed identical to the Worm-pep-predicted sequence, and cloned into a vector appropriate for eucaryotic expression. A [35S]guanosine 5'-O-(thiotriphosphate) (GTPgammaS) assay with membranes prepared from Chinese hamster ovary (CHO) cells transiently transfected with FLP15-R was used as a read-out for receptor activation. FLP15-R was activated by putative FLP15 peptides, GGPQGPLRF-NH2 (FLP15-1), RGPSGPLRF-NH2 (FLP15-2A), its des-Arg1 counterpart, GPSGPLRF-NH2 (FLP15-2B), and to a lesser extent, by a tobacco hornworm Manduca sexta FaRP, GNSFLRFNH2 (F7G) (potency ranking FLP15-2A > FLP15-1 > FLP15-2B >> F7G). FLP15-R activation was abolished in the transfected cells pretreated with pertussis toxin, suggesting a preferential receptor coupling to Gi/Go proteins. The functional expression of FLP15-R in mammalian cells was temperature-dependent. Either no stimulation or significantly lower ligand-evoked [35S]GTPgammaS binding was observed in membranes prepared from transfected FLP15-R/CHO cells cultured at 37 degrees C. However, a 37 to 28 degrees C temperature shift implemented 24 h post-transfection consistently resulted in an improved activation signal and was essential for detectable functional expression of FLP15-R in CHO cells. To our knowledge, the FLP15 receptor is only the second deorphanized C. elegans neuropeptide GPCR reported to date.

Natural variations of wild Caenorhabditis elegans isolates having either Phe-215 or Val-215 in NPR-1, a putative orphan neuropeptide Y-like G protein-coupled receptor, result in either "social" or "solitary" feeding behaviors (de Bono, M., and Bargmann, C. I. (1998) Cell 94, 679-689). We identified a nematode peptide, GLGPRPLRF-NH2 (AF9), as a ligand activating the cloned NPR-1 receptor heterologously expressed in mammalian cells. Shifting cell culture temperatures from 37 to 28 degrees C, implemented 24 h after transfections, was essential for detectable functional expression of NPR-1. AF9 treatments linked both cloned receptor variants to activation of Gi/Go proteins and cAMP inhibition, thus allowing for classification of NPR-1 as an inhibitory G protein-coupled receptor. The Val-215 receptor isoform displayed higher binding and functional activity than its Phe-215 counterpart. This finding parallels the in vivo observation of a more potent repression of social feeding by the npr-1 gene encoding the Val-215 form of the receptor, resulting in dispersing (solitary) animals. Since neuropeptide Y shows no sequence homology to AF9 and was functionally inactive at the cloned NPR-1, we propose to rename NPR-1 and refer to it as an AF9 receptor, AF9-R1.

KHEYLRF-NH(2) (AF2) is the most abundant FMRFamide-related peptide (FaRP) in Ascaris suum and also in many other parasitic and free-living nematodes. The AF2 abundance in the highly diverse nematodes and its potent and profound effects on the neuromuscular systems make AF2 and its receptor(s) very attractive targets for the discovery of novel broad-spectrum anthelmintics. Although FaRP receptors are believed to belong to the large family of G-protein coupled receptors (GPCRs), to date no AF2 receptor(s) have been cloned so there is no final proof to show that they are indeed G-protein coupled. In this study, using A. suum body wall muscle membranes, we showed that: (1) AF2 effectively (EC(50) 57 nM) induced a dose-dependent stimulation of [35S]GTP gamma S binding to the membranes, which is a hallmark of G-protein activation; (2) the high affinity binding of [125I-Tyr(4)]AF2 was inhibited in a dose-dependent manner by GTP with a K(i) of 10.5 nM (so-called guanine nucleotide effect, characteristic for GPCRs). Collectively, our results provide direct evidence for G-protein involvement in AF2-triggered receptor activation and thus confirm that the receptor for AF2 in A. suum is a GPCR.

Described in this report is a successful cloning and characterization of a functionally active Drosophila sulfakinin receptor designated DSK-R1. When expressed in mammalian cells, DSK-R1 was activated by a sulfated, Met(7-->Leu(7)-substituted analog of drosulfakinin-1, FDDY(SO(3)H)GHLRF-NH(2) ([Leu(7)]-DSK-1S). The interaction of [Leu(7)]-DSK-1S with DSK-R1 led to a dose-dependent intracellular calcium increase with an EC(50) in the low nanomolar range. The observed Ca(2+) signal predominantly resulted from activation of pertussis toxin (PTX)-insensitive signaling pathways pointing most likely to G(q/11) involvement in coupling to the activated receptor. The unsulfated [Leu(7)]-DSK-1 was ca. 3000-fold less potent than its sulfated counterpart which stresses the importance of the sulfate moiety for the biological activity of drosulfakinin. The DSK-R1 was specific for the insect sulfakinin since two related vertebrate sulfated peptides, human CCK-8 and gastrin-II, were found inactive when tested at concentrations up to 10(-5) M. To our knowledge, the cloned DSK-R1 receptor is the first functionally active Drosophila sulfakinin receptor reported to date., (©2002 Elsevier Science (USA).)

The type-A allatostatins A (AST-A) are a group of insect peptides with a common C-terminal motif Y/FXFGL-NH(2). The existence of at least four putative type A Drosophila melanogaster ASTs (called type A drostatins or DST-As) has been predicted from the sequence of a recently cloned DST-A preprohormone [C. Lenz et al. (2000) Biochem. Biophys. Res. Commun. 273, 126-1131]. SRPYSFGL-NH(2), (DST-3A), the only DST isolated from Drosophila so far, activated the first cloned DST-A GPCR (DAR-1) [N. Birgül et al. (1999) EMBO J. 18, 5892-5900]. A newly cloned orphan Dm GPCR, which shares 47% overall and 60% transmembrane region sequence identity with DAR-1, was classified as a second putative Dm DST-A receptor (DAR-2) [C. Lenz et al. (2000) Biochem. Biophys. Res. Commun. 273, 571-577]. Although activation of DAR-2 by DSTs has been postulated, no experimental evidence for that has been presented to date. In this study, we expressed both DAR-1 and DAR-2 in CHO cells and used a GTPgammaS and a Ca(2+) mobilization assay for pharmacological evaluation of the receptors. Synthetically prepared DST-As, as well as selected Diplotera punctata (cockroach) ASTs, activated DAR-1 and DAR-2 in both functional assays indicating ligand redundancy and cross species activity. Cell pretreatment with pertussis toxin led to some differences in the nature and magnitude of signaling pathways at the DAR-1 and DAR-2 receptors, suggesting possible differential coupling to cellular effector system(s) and distinct biological functions of each receptor in vivo., (Copyright 2001 Academic Press.)

Nervous systems of helminths are highly peptidergic. Species in the phylum Nematoda (roundworms) possess at least 50 FMRFamide-related peptides (FaRPs), with more yet to be identified. To date, few non-FaRP neuropeptides have been identified in these organisms, though evidence suggests that other families are present. FaRPergic systems have important functions in nematode neuromuscular control. In contrast, species in the phylum Platyhelminthes (flatworms) apparently utilize fewer FaRPs than do nematodes; those species examined possess one or two FaRPs. Other neuropeptides, such as neuropeptide F (NPF), play key roles in flatworm physiology. Although progress has been made in the characterization of FaRP pharmacology in helminths, much remains to be learned. Most studies on nematodes have been done with Ascaris suum because of its large size. However, thanks to the Caenorhabditis elegans genome project, we know most about the FaRP complement of this free-living animal. That essentially all C. elegans FaRPs are active on at least one A. suum neuromuscular system argues for conservation of ligand-receptor recognition features among the Nematoda. Structure-activity studies on nematode FaRPs have revealed that structure-activity relationship (SAR) "rules" differ considerably among the FaRPs. Second messenger studies, along with experiments on ionic dependence and anatomical requirements for activity, reveal that FaRPs act through many different mechanisms. Platyhelminth FaRPs are myoexcitatory, and no evidence exists of multiple FaRP receptors in flatworms. Interestingly, there are examples of cross-phylum activity, with some nematode FaRPs being active on flatworm muscle. The extent to which other invertebrate FaRPs show cross-phylum activity remains to be determined. How FaRPergic nerves contribute to the control of behavior in helminths, and are integrated with non-neuropeptidergic systems, also remains to be elucidated.

An autolysis-resistant mutant of the HIV-I protease was employed for removal of metabolically stabilized and highly bioactive analogues of bovine growth-hormone-releasing factor (bGRF) from their larger either synthetic or recombinant precursors. The N-terminal four amino acids in two selected model GRF analogues, Y1IDAIFTSSYRKVLAQLSARKLLQDILSRQVF32-OH (I; GRF32) and Y1IDAIFTSSYRKVLAQLSARKLLQDILSRQ30-OH (IA; GRF30), conform well to the specificity of the HIV-I protease for residues in the P1' to P4' positions of its peptide substrates. A variety of amino acids were tried in the N-terminal extension (positions P4-P1) to fit the protease substrate specificity for the 8 amino acids in positions P4-P4'. A synthetic precursor of I, extended N-terminally with RQVF-, a sequence representing the four C-terminal residues in I, was effectively cleaved by the protease at the Phe-1-Tyr1 bond (... RQVF-decreases-YIDA ...) to release GRF32. However, when several soluble fusion proteins linked to GRF32 by the RQVF sequence were expressed in Escherichia coli, attempts to cleave out the core GRF32 met with variable, and only limited, success. By random mutagenesis in a propeptide segment, [MGQSVAQVF]-decreases-GRF30, (II) was identified as a construct that showed reasonably high-level expression in E. coli and was effectively processed by the HIV-I protease. A yield of 5 mg of pure GRF30 was obtained/litre of culture medium after a single HPLC purification step.

PF4 has previously been shown to have potent inhibitory effects on myoactivity of somatic muscle strips from the nematode. Ascaris suum. This study examined the bioactivity and metabolic stability of position 2- and position 5-modified analogues of PF4. Although the analogues [Leu5]PF4,[Ala2]PF4, [Gly2]PF4, [Ala2,Leu5]PF4, and [Gly2,Leu5]PF4 all had qualitatively similar inhibitory effects on A. suum somatic muscle strips, their effects were quantitatively distinguishable and had the order of potency: PF4 = [Leu5]PF4 > > [Ala2]PF4 = [Ala2,Leu5]PF4 > > [Gly2]PF4 = [Gly2,Leu5]PF4, Leu5 for Ile5 substitutions in PF4 did not alter the activity of this peptide: however, Gly2/Ala2 for Pro2 substitutions reduced, but did not abolish, peptide activity. Peptide stability studies revealed that [Gly2]PF4(2-7) and -(3-7) and [Ala2]PF4(2-7), -(3-7), and -(4-7) fragments were generated following exposure to A. suum somatic muscle strips. However, the parent peptide (PF4) was not metabolized and appeared to be resistant to the sequential cleavages of native aminopeptidases. Observed analogue metabolism appeared to be due to the activity of released aminopeptidases as identical fragments were generated by incubation in medium that had been exposed to somatic muscle strips and from which the strips had been removed prior to peptide addition. It was found that the muscle stretching and bath mixing characteristics of the tension assay led to more effective release of soluble enzymes from muscle strips and thus greater peptide degradation. These studies reveal that Pro2 in PF4 is not essential for the biological activity of this peptide; however, it does render the peptide resistant to the actions of native nematode aminopeptidases.

A novel FMRFamide-related heptapeptide, Lys-Pro-Asn-Phe-Ile-Arg-Phe-NH2 (KPNFIRFamide), was isolated and characterized from acid ethanol extracts of the free-living nematode, Panagrellus redivivus. Whole-worm extracts contained > or = 9 pmol KPNFIRFamide/g wet weight. A synthetic replicate of this peptide induced a rapid relaxation of tone and inhibited spontaneous contractility in isolated innervated and denervated body-wall muscle strips of the parasitic nematode, Ascaris suum. KPNFIRFamide (0.1 nM) induced measurable relaxations in 50% of the muscle preparations examined. Concentrations > or = 0.3 nM induced relaxation in 100% of muscle preparations examined. The relaxation was short-lived at concentrations of peptide > or = 1 microM and displayed a profile typical of receptor desensitization. These data suggest the occurrence of a closely related peptide in A. suum and add further evidence to the concept of primary structural conservation of FaRPs within the nematodes.

The presence of Val2 in mGRF(1-42)OH is unique and, as shown in this study, renders this GRF resistant to plasma DPP-IV, the main enzyme responsible for rapid hydrolysis and inactivation of Ala2-containing GRFs from other species via cleavages between Ala2-Asp3. The presence of DPP-IV activity in mouse serum, and mouse and bovine plasma has been demonstrated with Gly-Pro-p-nitroanilide and/or with two DPP-IV-sensitive bGRF analogs, [Leu27]bGRF(1-29)NH2 and [Ala15,Leu27]bGRF(1-29)NH2, which were effectively converted to their respective (3-29) fragments. During incubations of mGRF(1-42)OH in mouse serum or plasma, as well as in bovine plasma in vitro, no major fragments were detectable, except for small amounts of metabolites with HPLC retention times corresponding to those of mGRF(12-42)OH and mGRF(21-42)OH, indicative of possible trypsin-like cleavages between Arg11-Lys12 and Arg20-Lys21. Both mGRF(1-42)OH (t1/2 52-78.5 min) and [Val2,Ala15,Leu27]-bGRF(1-29)NH2 (t1/2 78.5 min) disappeared 5 to 7 times faster in mouse than in bovine plasma, indicating much higher activity of various degrading enzymes in mouse plasma. In summary, our data provide evidence that mGRF(1-42)OH, despite its resistance to plasma DPP-IV, is degraded relatively fast in mouse plasma or serum because of trypsin-like and other, non-DPP-IV-related, proteolytic cleavages.

Ion-spray ionization mass spectrometry with gentle conditions for solvent removal has been reported as a useful tool for detection of high-affinity noncovalent complexes of biological relevance formed in solution. Two main objectives of this study were (i) to find whether other types of electrospray ionization (ESI) sources, e.g. where the solvent is removed with the help of heat (thermally assisted electrospray), could be utilized for detection of noncovalent biological complexes of high and low affinity and (ii) to find whether ESI-MS can be used for detection of the association of bovine serum albumin (BSA) with biologically active peptides. Using a well-defined high-affinity association of FK506 with its binding protein (FKBP) as model system we proved that ESI-MS with thermally assisted interphase can be used for detection of the FK506-FKBP complexes in a similar way as was previously shown for electrospray mass spectrometry (Ganem et al., J. Am. Chem. Soc. 113, 6294 (1991)). In mixtures of BSA with a 9-10 molar excess of biologically active peptides, such as growth hormone releasing factor (GRF), glucagon, bradykinin or insulin in ammonium acetate at pH 7.5, complexes with a ratio of 1:1, 1:2 and in some cases 1:3 were detected. On the other hand, these complexes disappeared upon acidification, pointing to their noncovalent nature.(ABSTRACT TRUNCATED AT 250 WORDS)

The objective of this study was to determine whether the rates of deamidation of Asn8 in selected growth hormone releasing factor (GRF) analogs were related to the peptide's secondary structures in solution. Bovine or human [Leu27]GRF(1-32)NH2 (both having Gly at position 15), [Ala15Leu27]bGRF(1-32)NH2 and [Pro15Leu27]bGRF(1-32)NH2 were used as model peptides. The peptide helical content (assessed by CD) increased with the increasing methanol concentration and was as follows: 7, 12 and 18% in 0% MeOH; 24, 48 and 52% in 40% MeOH; and 41, 77 and 81% in 80% MeOH for Pro15Leu27 bGRF(1-32)NH2, [Leu27]hGRF(1-32)NH2 and Ala15Leu27 bGRF(1-32)NH2, respectively. 2D NMR studies done in the presence of 40% CD3OH indicated more helical structure for the Ala15 analog as compared to [Leu27]hGRF(1-32)NH2. In both these peptides Asn8 was included in the helical region. In contrast, the lack of conformational information for the Pro15 analog indicated little helical structure around Asn8. The peptides' deamidation rates decreased and their half-lives increased with increasing MeOH concentrations. At 40% MeOH, the least helical Pro15 bGRF analog (t1/2 = 10.78 h) deamidated 1.5 and 2 times faster than its Gly15 (t1/2 = 15.74 h) and Ala15 (t1/2 = 21.53 h) counterparts, respectively. This study indicates that helical environment around Asn8 in GRF makes this residue less prone to deamidation.

The literature reported DPP-IV substrate specificity includes oligopeptides with a penultimate Pro, Hyp or Ala residue. Bovine GRF is a substrate for DPP-IV and is rapidly degraded by the enzyme via removal of its N-terminal Tyr-Ala. Incubation of selected GRF analogs from the [X2,Ala15,Leu27]bGRF(1-29)NH2 series with a porcine-kidney-derived DPP-IV in PBS (pH 7.4) resulted in cleavage at the X2-Asp3 bond. The extent of enzymatic hydrolysis varied with X2 as reflected in the following relative cleavage rates: Ala2 (100%), Ser2 (4%), Thr2 (2.5%), Val2 (0.53%), Ile2 (0%). These cleavages were sequestered when similar experiments were performed in the presence of the DPP-IV-specific inhibitor N-epsilon-(p-NO2-benzyloxycarbonyl)-Lys-Pro-OH. A side reaction, buffer-induced deamidation of Asn8, contributed less than 5% of the total substrate degradation. Although our finding qualitatively extends the DPP-IV substrate specificity to also include N-terminal X-Ser, X-Thr and X-Val sequences, quantitatively, relatively fast cleavages of the GRFs with Ala2 make the latter preferred substrates for DPP-IV. The data presented here indicates that the observed GRF(3-29) fragment formation upon incubation of Ser2- and Thr2-substituted bGRF analogs in bovine plasma could have been DPP-IV-related.

In order to prepare GRF analogs with high activity in vivo, a strategy was undertaken to stabilize the peptide to dipeptidylpeptidase IV (DPP-IV), a protease found in plasma which inactivates native human and bovine GRF by cleavage of the Ala2-Asp3 bond. Replacement of the Ala2 residue with Ser, Thr, or Gly in [Leu27]bGRF(1-29)NH2 resulted in peptides greatly stabilized against proteolysis in plasma, but having low inherent GH-releasing activity when tested in bovine pituitary cell cultures. Replacement of Gly15 with Ala15 was marginally effective in improving the in vitro bioactivity of this group of peptides. When tested for GH-hormone release in steers, however, the Thr2,Ala15 analog was four times more potent than bGRF(1-44)NH2. Eleven additional analogs from the [X2,Ala15,Leu27]bGRF(1-29)NH2 series were synthesized and evaluated for metabolic stability in bovine plasma and for GH releasing activity in steers in vivo and in rat pituitary cells in vitro. Two compounds, [Val2,Ala15,Leu27]dGRF(1-29)NH2 and [Ile2,Ala15,Leu27]-bGRF(1-29)NH2, had increased GH-releasing activity in steers over that of [Thr2,Ala15,Leu27]-bGRF(1-29)NH2 and over a previously reported super-potent analog, [desNH2Tyr1,D-Ala2,Ala15]-hGRF(1-29)NH2.

Relative growth hormone-releasing potencies of bovine growth hormone-releasing factor (bGRF) analogs bGRF(1-44)NH2 (I), Leu27-bGRF(1-29)NH2 (II) and D-Ala2, Leu27-bGRF(1-29)NH2 (III) in in vitro bovine anterior pituitary cell cultures were determined to be 100%, 48% and 77%, respectively. The potencies of II and III, although numerically different, were not statistically different. Leu27-bGRF(3-29)NH2 (IV) was approximately 10,000 times less potent than 1. 1H NMR studies of peptides II, III and IV in 35% d3-2,2,2-trifluorethanol (TFE)/65% phosphate buffer at pH 4 revealed very similar, highly helical secondary structures in the 8-29 region, with only subtle differences at the N-termini. This lack of correlation between secondary structure in solution and in vitro bioactivity suggests that either 1) the biological conformations induced at the GRF receptor for II and III vs. IV are different from those generated in TFE/buffer, 2) similar secondary structures may be necessary but not sufficient for the observed bioactivity or 3) residues 1 and 2 of analogs II and III are important contact residues crucial for effective GRF-receptor interaction.

A bovine growth hormone-releasing factor analog, Leu27-bGRF(1-29)NH2, was rapidly hydrolyzed to Leu27-bGRF(3-29)NH2 when incubated at 0.03 mM with porcine and bovine plasma at 37 degrees C in vitro (t1/2 = 8.4 min and 22.1 min, respectively). The site of cleavage was the same as that reported by Frohman et al. (J. Clin. Invest. 78, 906-913, 1986) for the GRF/human plasma system and was suggested by the authors to be due to the presence of dipeptidylpeptidase IV (DPP-IV) in human plasma. The DPP-IV-like activity of porcine plasma, determined with Gly-Pro-p-nitroanilide as substrate at pH 7.6 was about 2- to 3-fold higher than that of bovine plasma and seems to correlate well with the more rapid degradation of the GRF analog in porcine plasma. The hormone half-life was extended to 83.3 min when Leu27-bGRF(1-29)NH2 was incubated in vitro with bovine plasma in the presence of an equimolar amount of diprotin A (a competitive DPP-IV inhibitor). Dipeptidylpeptidase II-like activity of porcine and bovine plasma (which may overlap with substrate specificity of DPP-IV) was measured with Lys-Ala-beta-naphthylamide and at pH 7.6 was found to be relatively low (3% and 21% of the corresponding plasma DPP-IV activities). Tyr-beta-naphthylamide was hydrolyzed slowly by porcine plasma and not degraded at all by bovine plasma, which suggests that the sequential cleavage from the GRF N-terminus starting with Tyr at position 1 is not dominant.(ABSTRACT TRUNCATED AT 250 WORDS)