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J R Reeve

Publications and source records attributed to J R Reeve.

16 recordsLinked to original sources
PubMed1992

Novel generation of hormone receptor specificity by amino terminal processing of peptide YY.

The physiological significance of multiple Y receptors has not been determined since until recently only one form of endogenous agonists was known, namely PYY1-36 and NPY1-36. Recently, a new molecular form of PYY was characterized as des(Tyr-Pro)PYY (PYY3-36 or PYY-II). Its ability to interact at various Y receptors was not characterized. Analytical chromatography of fresh canine colon extracts shows two peaks of immunoreactivity eluting in the positions of PYY-II and PYY1-36 (PYY). PYY-II was about 40% of the total PYY immunoreactivity indicating that it is one of the major forms of PYY expressing its biological activity. It is shown that PYY-II will not displace label from the Y1 receptors found on a human neuroblastoma cell line. It is further shown that PYY-II is as potent as PYY for the inhibition of pancreatic secretion, which must occur through Y2 receptors. The enzymatic removal of Tyr-Pro from PYY to form PYY-II must therefore regulate the relative expression of a non-selective agonist (PYY) to a highly selective Y2 agonist (PYY-II). Amino terminal processing of PYY represents a novel type of regulation of peptide hormone specificity. It has important biological implications for PYY and potential relevance for other peptide hormone receptor systems.

Animals↗
PubMed1992

Gastrin-releasing peptide (GRP) is not mammalian bombesin. Identification and molecular cloning of a true amphibian GRP distinct from amphibian bombesin in Bombina orientalis.

On the basis of structural homology and similar biological activity, gastrin-releasing peptide (GRP) has been considered the mammalian equivalent of amphibian bombesin. In this paper we now show this to be incorrect. Chromatography of frog (Bombina orientalis) gut extracts demonstrated two peaks of bombesin-like immunoreactivity (BLI), one similar in size to GRP and one similar in size to amphibian bombesin. These peaks were purified by high pressure liquid chromatography then subjected to mass spectrometric analyses to determine molecular weights and amino acid sequence. Based on the amino acid sequence of the lower molecular weight BLI species, a mixed oligonucleotide probe was prepared and used to screen a B. orientalis stomach cDNA library. Sequence analysis showed that all hybridizing clones encoded a 155-amino acid protein homologous to the mammalian GRP precursor. The mass spectra of the high and low molecular weight peaks of frog gut BLI were consistent with their origin from the processing of the frog GRP (fGRP) precursor into GRP-29 and GRP-10, just like the processing of the rat GRP precursor. Sequence homology showed that the fGRP precursor is more homology showed that the fGRP precursor is more closely related to the mammalian GRP precursors than to either the frog bombesin or frog ranatensin precursors. Northern blot analysis showed that fGRP is encoded by a mRNA of 980 bases, clearly different from the 750-base mRNA which encodes frog bombesin. Northern blot analysis and in situ hybridization showed fGRP mRNA in frog brain and stomach and bombesin mRNA in frog skin, brain, and stomach. That frogs have independent genes for both GRP and bombesin raises the possibility that mammals have an as yet uncharacterized gene encoding a true mammalian bombesin.

Amino Acid Sequence↗
PubMed1991

Characterization of canine intestinal cholecystokinin-58 lacking its carboxyl-terminal nonapeptide. Evidence for similar post-translational processing in brain and gut.

An antibody raised against a synthetic cholecystokinin (CCK) analog, (1-27)-(CCK)-33, corresponding to the midregion of CCK-58, detected immunoreactivity in intestinal extracts which eluted between the positions of CCK-33/39 and CCK-58 on high performance liquid chromatography. This peak, lacking carboxyl-terminal cholecystokinin immunoreactivity, was purified by reverse phase and cation-exchange chromatographies. Amino acid, mass spectral, and microsequence analysis established that it was the amino-terminal desnonapeptide fragment of cholecystokinin-58, (1-49)-CCK-58. It was demonstrated further that CCK-58 has less biological activity than CCK-8, suggesting that the amino terminus either sterically hindered the ability of CCK-58 to exert its biological activity or that its amino terminus acted at another site to inhibit release of amylase from rat pancreatic acini. The desnonapeptide of CCK-58 by itself had no biological activity, nor did it affect CCK-8-stimulated amylase release from isolated rat pancreatic acini, suggesting that the amino terminus shields the carboxyl terminus from expressing its biological activity. Its presence in intestine suggests that it is released into the circulation where it could be detected by midregion antibodies. The presence of high proportions of (1-49)-CCK-58 indicates that most CCK-8 is directly derived from CCK-58. Its occurrence in brain and intestine indicates similar processing for procholecystokinin in both tissues.

Amino Acid Sequence↗
PubMed1991

Purification and structural characterization of progastrin-derived peptides from a human gastrinoma.

Several peptides derived from the gastrin-predicted preprohormone sequence were isolated from a human gastrinoma by gel permeation, anion exchange, and reverse phase chromatography. The peptides were identified and characterized structurally by a combination of radioimmunoassays, mass spectral analysis, and microsequence analysis. The largest peptide, progastrin-(1-35) (cryptagastrin), extends from the putative processing site for the signal peptidase to the double basic residues adjacent to the amino terminus of gastrin 34. A shorter form of this peptide, progastrin-(6-35) (cryptagastrin-(6-35), was also isolated in smaller amounts. In addition, sulfated and nonsulfated gastrin 17 amides (progastrin-(55-71)) and the glycine-extended nonsulfated gastrin 17 (progastrin-(55-72)) were identified by radioimmunoassay, and their structures were confirmed by mass spectral analysis. Isolation of cryptagastrin indicates that the signal peptide of human preprogastrin contains 21 amino acid residues, and progastrin, therefore, contains 80 amino acids. There is minimal processing of the cryptic peptide preceding the sequence of gastrin 34. An amidated gastrin form larger than gastrin 34 could contain 71 amino acids. No evidence was obtained for processing that would produce gastrins containing more than 34 but less than 71 amino acid residues.

Amino Acid Sequence↗
PubMed1991

Development of a biotinylated analog of substance P for use as a receptor probe.

The development of a biotinylated substance P (SP) analog for use as a receptor probe is reported. The lysine in position 3 of SP was substituted by arginine and an amino terminal extension (NTE-SP) was added consisting of Lys-Tyr-Gly-Gly-Gly-Gly-Gly-Gly. Biotinylation of the N-terminal lysine was performed. The biotinylated peptide was purified by high performance liquid chromatography and characterized by mass spectral analysis. Binding studies using human IM-9 lymphoblasts with the biotinylated SP analog (biotin-NTE[Arg3]SP) and native SP yielded dissociation curves which were identical. In addition, the biotinylated SP analog retained functional activity similar to that of native SP in altering intracellular calcium concentration of Fura-2 loaded isolated rabbit colonic myocytes. Applicability of the SP receptor probe was demonstrated by using the streptavidin-peroxidase detection system to identify SP receptors on human IM-9 lymphoblasts. In conclusion, a biotinylated SP analog has been developed which retains the functional characteristics of the native peptide and is a useful and versatile probe for receptor studies.

Amino Acid Sequence↗
PubMed1990

Biological activity of a bombesin-like peptide extracted from the intestine of the ratfish, Hydrolagus colliei.

1. Ratfish (Hydrolagus colliei) intestines were boiled in water to inactivate proteases and then treated with cold 4% trifluoroacetic acid to extract bombesin-like peptides. 2. The extract was fractionated in several steps using reverse-phase and ion exchange HPLC, and bombesin-like immunoreactive peptides were detected by radioimmunoassay using an antiserum specific for the bioactive C-terminal region of bombesin. 3. A highly purified bombesin-like peptide-containing fraction stimulated amylase release in a dose-responsive fashion from rat pancreatic acini; the dose-response curve was parallel to a bombesin standard, and the ratfish peptide stimulated the same maximal rate of amylase secretion as the bombesin standard. 4. A potent, highly selective bombesin receptor antagonist completely abolished the stimulation of amylase release caused by the ratfish peptide, demonstrating the specificity of the response. 5. Estimates of the bombesin-like peptide concentration of this fraction by radioimmunoassay and by bioassay were nearly identical, indicating that ratfish bombesin is very similar biologically and antigenically to frog skin bombesin.

Amylases↗
PubMed1990

Cloning of cDNAs encoding amphibian bombesin: evidence for the relationship between bombesin and gastrin-releasing peptide.

Bombesin is a tetradecapeptide originally isolated from frog skin; its mammalian homologue is the 27-amino acid peptide gastrin-releasing peptide (GRP). cDNAs encoding GRP have been cloned from diverse species, but little is yet known about the amphibian bombesin precursor. Mass spectrometry of HPLC-separated skin exudate from Bombina orientalis was performed to demonstrate the existence of authentic bombesin in the skin of this frog. A cDNA library was prepared from the skin of B. orientalis and mixed oligonucleotide probes were used to isolate cDNAs encoding amphibian bombesin. Sequence analysis revealed that bombesin is encoded in a 119-amino acid prohormone. The carboxyl terminus of bombesin is flanked by two basic amino acids; the amino terminus is not flanked by basic amino acids but is flanked by a chymotryptic-like cleavage site. Northern blot analysis demonstrated similarly sized bombesin mRNAs in frog skin, brain, and stomach. Polymerase chain reaction was used to show that the skin and gut bombesin mRNAs encoded the identical prohormones. Prohormone processing, however, differed between skin and gut. Chromatography showed the presence of only authentic bombesin in skin whereas gut extracts contained two peaks of bombesin immunoreactivity, one consistent in size with bombesin and one closer in size to mammalian GRP. Thus the same bombesin prohormone is processed solely to bombesin in skin but is processed to a peptide similar in size to bombesin and to a peptide similar in size to mammalian GRP in stomach.

Amino Acid Sequence↗
PubMed1990

In-vitro production of precursor peptides for thyrotropin-releasing hormone by human semen.

Thyrotrophin-releasing hormone (TRH) and related peptides occur in high concentrations in human semen. TRH derives from a 242-amino acid precursor protein, prepro-TRH, with six repetitive sequences of -Lys-Arg-Gln-His-Pro-Gly-Lys/Arg)-Arg- connected by hydrophobic linking sequences. Antibodies to TRH-Gly (pGlu-His-Pro-Gly), a final precursor for TRH formation, were used to detect this tetrapeptide as well as other prepro-TRH fragments which cross-react with these antibodies. The total TRH-Gly immunoreactivity decreased significantly after vasectomy. The TRH-Gly immunoreactivity in semen increased significantly during in-vitro incubation at 0 or 37 degrees C, to a peak value at 5 h, followed by an exponential decline, with t 1/2 equal to 11 h at 37 degrees C. At 60 degrees C, however, the TRH-Gly immunoreactivity rose continuously, attaining, after 20 h, a level 2.2 times that at the start of the incubation (P less than 0.001). Reversed-phase high pressure liquid chromatography (HPLC) revealed both hydrophobic and hydrophilic TRH-Gly immunoreactive peptides in semen with both classes of peptides increasing significantly with heating to 60 degrees C. Cation exchange chromatography of pooled human semen incubated at 60 degrees C revealed a 4.3-fold increase in a TRH-Gly immunoreactive peak which co-eluted with synthetic TRH-Gly, and a 30% increase in another TRH-Gly immunoreactive peak identified as Glu-His-Pro-Gly. A minor, TRH-Gly immunoreactive peak increased 50-fold (P less than 0.001) during 20 h at 60 degrees C. This material co-eluted with Arg-Gln-His-Pro-Gly which is formed by enzymic cleavage of the paired basic residues flanking this sequence in prepro-TRH. When synthetic Arg-Gln-His-Pro-Gly was incubated with fresh semen at 60 degrees C a rapid conversion of most of this peptide to Glu-His-Pro-Gly, Gln-His-Pro-Gly and TRH-Gly occurred within 30 min. These data are consistent with thermal inactivation of the amidation and degrading enzymes at 60 degrees C while the trypsin-like enzymes which cleave the precursor peptide at the paired basic residues remain relatively unaffected. Because other investigators have found the C-terminal amidating enzymes to be associated with secretory vesicles and to be co-secreted with the vesicular contents, we suggest that secretory epithelia of the male reproductive system secrete TRH and TRH-related precursor peptides along with the alpha-amidating enzymes which continue processing of prepro-TRH in the post-ejaculatory seminal fluid.

Amino Acid Sequence↗
PubMed1990

Characterization of the major form of cholecystokinin in human intestine: CCK-58.

Acid extracts of human intestines obtained from surgical samples or from organ donors contain cholecystokinin (CCK) immunoreactivity. From surgical samples, extracted and eluted quickly, greater than 75% of the CCK immunoreactivity eluted in the same region as purified canine CCK-58 during analytical reverse-phase high-pressure liquid chromatography (HPLC). A major portion of the CCK immunoreactivity from donor intestinal extracts also eluted in this region. This immunoreactivity has been purified from human intestinal extracts by a series of several reverse-phase and cation-exchange chromatographies. Amino acid and microsequence analysis showed that this immunoreactivity is human CCK-58. Tryptic digestion of purified human CCK-58 produced another immunoreactive form that eluted in the position of CCK-8 during analytical reverse-phase HPLC. The immunoreactivity of the trypsin-digested material was 2.6-fold higher than that of an identical sample of CCK-58 incubated without trypsin. Thus the carboxyl-terminal antibody used for radioimmunoassay cross-reacts greater than twofold less with human CCK-58. This diminished cross-reactivity would lead to an underestimation of the relative proportions of CCK-58 in tissue and plasma extracts. If CCK-58 is the major circulating form this diminished cross-reactivity would also lead to underestimations of the circulating levels of total CCK. Determination of human CCK-58 structure confirms that one of the major components of human CCK that expresses biological activity is CCK-58.

Amino Acid Sequence↗
PubMed1976

Studies on the disulfide bonds of glycoprotein hormones. Course of reduction of bovine luteinizing hormone, bovine thyroid-stimulating hormone, and their subunits.

The five disulfide bonds of isolated alpha subunits of luteinizing hormone (LH) and thyroid-stimulating hormone (TSH) are completely reduced at pH 8.5 in 15 min with no denaturant required and with only a slight excess of reducing agent. At pH 7.0, reduction is complete after 6 to 10 h. These results together with an earlier study concerning the positions of the two most readily reduced bonds (Cornell J.S., and Pierce, J.G. (1974) J. Biol. Chem. 249, 4166-4174) show that, in the isolated alpha subunit, all disulfides are readily accessible, although it is possible that a change in conformation, after rapid initial reduction of two disulfides, makes the remaining three more susceptible to reduction. No partially reduced and S-carboxymethylated intermediates were found at pH 7.0 other than those seen at pH 8.5, nor were additional intermediates found at pH 8.5 when reduction was initiated in the presence of alkylating agent. In contrast, reduction of the beta, hormone-specific, subunits of LH and TSH, while complete at pH 8.5 after 2 to 6 h, does not proceed to completion at pH 7.0 even after 24 h or upon addition of 6 M urea or large concentrations of reducing agent, and partially reduced intermediates useful in location of disulfide bridges can be trapped (e.g. Reeve, J.R., Cheng, K.-W., and Pierce, J.G. (1975) Biochem. Biophys. Res. Commun. 67, 149-155). Little or no reduction of the intact hormones is found at pH 7.0 in the absence of denaturing agents. This protection by the intact structure shows that the two most readily reduced disulfides of the alpha subunit and the single most readily reduced sidulfide of the beta subunits are either in regions of subunit-subunit contact or that these bonds become more reactive in the isolated subunits because of different influences by neighboring groups. At pH 8.5, intact LH is completely reduced after 6 h, but intact TSH is more resistant to reduction, which may reflect a higher affinity between subunits than exists in LH.

Animals↗
PubMed1976

Structure and structure-function relationships in glycoprotein hormones.

Relationships between the sequences of thyrotropin (thyroid-stimulating hormone, TSH), lutropin (luteinizing hormone, LH), human choriogonadotropin (chorionic gonadotropin, hCG) and follitropin (follicle-stimulating hormone, FSH) are now well established. Each beta-subunit contains six disulphide bonds and considerable homology is seen when all four linear sequences are aligned with half-cystine residues in juxtaposition. Major questions about the tertiary structures of the subunits and their interactions to form active hormone remain. Determination of the disulphide bridges in both alpha- and beta-subunits has not yielded to usual methods and conflicting data about the alpha-subunit have been reported. Partial reduction of the beta-subunits of LH and TSH with subsequent labelling of the cysteines formed has shown that a single bond is first reduced. This bond is between positions 93 and 100 in LH-beta and the corresponding positions 88-95 in TSH-beta. Thus, as would be expected from the fact that interhormone hybrids can be made with the common alpha-subunits, the chemical data, though still limited, indicate similar tertiary structures for the different beta-subunits. To investigate whether other useful intermediates can be obtained after partial reduction, we have studied reduction and derivative formation in various conditions. Intact LH is more resistant to reduction than either its alpha- or beta-subunit but no intermediates have been observed which are not present after partial reduction of individual subunits. Preliminary experiments on the reoxidation of fully reduced alpha-subunit show that the reoxidized material will recombine with native beta-subunits to yield biologically active TSH or LH. Studies from this and other laboratories on chemical modifications of several amino acid residues of glycoprotein hormones and their subunits are also summarized.

Amino Acid Sequence↗
PubMedDate not supplied

Structural characterization of calcitonin gene-related peptide purified from rabbit intestine.

Calcitonin gene-related peptide (CGRP) immunoreactive material has been found in extracts of the intestine, however, the structure of intestinal CGRP is not known. Analytical reverse phase HPLC and ion-exchange FPLC revealed one predominant immunoreactive CGRP peak in rabbit intestinal extracts. This material was purified from rabbit intestine by sequential steps of reverse phase HPLC and ion-exchange FPLC. Microsequence and mass spectral analysis of the purified peptide and its chymotryptic fragments were consistent with the structure: GCNTATCVTHRLAGLLSRSGGMVKSNFVPTNVGSEAF-amide. Rabbit intestinal CGRP is identical to human CGRP-II in 35 of 37 amino acid residues. Two amino acid differences were detected at position 1, with Gly in rabbit CGRP instead of Ala in human CGRP-II, and at position 35, with Glu instead of Lys, respectively. Rabbit CGRP differed from human CGRP-I by three additional amino acids at positions 3, 22, and 25. This report shows that a CGRP form which closely resembles human CGRP-II, by means of chemical characterization, is the predominant form in rabbit intestine. Rabbit CGRP is the only CGRP form which has Gly as the amino terminal amino acid. Since the amino terminus of CGRP seems to be important for expression of bioactivity, the biological activity of rabbit CGRP may differ from human, rat and porcine CGRP.

Amino Acid Sequence↗
PubMedDate not supplied

Biotinylation of a bombesin/gastrin-releasing peptide analogue for use as a receptor probe.

The development of a biotinylated bombesin/gastrin-releasing peptide (GRP) for use as a receptor probe is reported. The lysine13 of a GRP-27 was substituted by arginine and lysine was added to the amino terminus. Biotinylation of the N-terminal lysine was performed. The biotinylated peptide was purified by HPLC and characterized by mass spectral analysis. Binding studies with murine Swiss 3T3 fibroblasts, cells known to express bombesin/GRP receptors, yielded a dissociation curve for the biotinylated GRP-27 analogue (biotin-Lysyl[Asp12,Arg13]GRP-27) which was nearly identical to that of native GRP. Using studies of gastrin release from isolated canine G cells, equipotent functional activity of the biotinylated probe and unmodified GRP was demonstrated. Measurements of retained 125I-avidin confirmed that the biotin/avidin interaction could occur once the biotin-peptide complex was bound. Applicability of the probe was demonstrated with fluorescent microscopy using avidin-FITC on Swiss 3T3 fibroblasts. In conclusion, a novel biotinylated bombesin/GRP analogue has been developed which retains the functional characteristics of the native peptide and is a useful probe for receptor studies.

Amino Acid Sequence↗
PubMedDate not supplied

Isolation of the neuropeptide SALMFamide-1 from starfish using a new antiserum.

We have raised antisera in rabbits to a conjugate of thyroglobulin and Lys-Tyr-Ser-Ala-Leu-Met-Phe-NH2 (KYSALMFamide), a synthetic analog of the starfish neuropeptide S1 (Gly-Phe-Asn-Ser-Ala-Leu-Met- Phe-NH2). The sensitivity and specificity of two antisera (BL and SL) for S1 were established by testing the ability of S1 and structurally related peptides (SALMFamide-2 and various FMRFamide-related peptides) to displace iodinated KYSALMFamide from the serum antibodies in an RIA. Both antisera are sensitive to femtomolar amounts of S1. BL is highly specific for S1 but SL is not, since it is also able to detect femtomolar amounts of the FMRFamide-related peptides. We have used the BL antiserum in the RIA to monitor the purification of S1 immunoreactivity from radial nerve cord extracts of both Asterias rubens and Pycnopodia helianthoides. The partial amino acid sequence GFNSALM was obtained from automated Edman degradation sequencing of pure immunoreactive peaks from both species.

Amino Acid Sequence↗