60 YEARS OF POMC: Lipotropin and beta-endorphin: a perspective.

Many important fields of research had a humble origin. In the distant past, A J P Martin's discovery that amino acids could be separated by paper chromatography and Moore and Stein's use of columns for quantitative amino acid analysis provided the first steps towards the determination of structure in complex biologically active molecules. They opened the door to reveal the essential relationship that exists between structure and function. In molecular endocrinology, for example, striking advances have been made by chemists with their expertise in the identification of structure working with biologists who contributed valuable knowledge and experience. Advantage was gained from the convergence of different background, and it is notable that the whole is greater than the sum. In the determination of structure, it may be recalled that four of the world's great pioneers (Archibald Martin, Rodney Porter, Fred Sanger and Vincent du Vigneaud) were acknowledged for their fundamental contributions when individually they were awarded the Nobel Prize. They foresaw that the identification of structure would prove of outstanding importance in the future. Indeed, study of the structures of ß-endorphin and enkephalin and the different forms of opiate activity they engender has led to a transformation in our understanding of chemical transmission in the brain.

Characterization of neutral TRH-like peptides in mammary gland, mammary tumors and milk.

Three pyroglutamylpeptide amides, pGlu-Glu-Pro amide, pGlu-Phe-Pro amide and pGlu-Gln-Pro amide, with similar structures to thyrotropin-releasing hormone (TRH), have been identified previously in the male reproductive system. We report here that rat and human mammary gland contain neutral TRH-immunoreactive peptides which are not retained on cation or anion exchange chromatography and that similar peptides occur in the milk of rat, cow, ewe and sow. The TRH-like peptides in lyophilized milk from the cow were purified by gel exclusion chromatography, mini-column cation exchange chromatography and reversed phase high performance liquid chromatography (HPLC) and the chromatographed peptides were located by TRH radioimmunoassay (RIA). In each chromatographic system the major TRH-immunoreactive peptide from cow milk exhibited identical behavior to pGlu-Phe-Pro amide; in addition there were two minor TRH-immunoreactive components. The possible physiological role of the TRH-like peptides in the mammary gland is discussed. In a series of patients with breast carcinoma, mammary tumor tissue was shown to contain approximately four times more TRH-like peptide than normal mammary tissue from the same patient, raising the possibility that the TRH-like peptides may be implicated in tumor development.

Protirelin (thyrotropin-releasing hormone) in thyroid gland: possible involvement in regulation of thyroid status.

AIM: To establish the presence of the hypothalamic hormone protirelin (thyrotropin-releasing hormone, TRH) in human thyroid and to investigate whether the concentration of this peptide in the thyroid gland is sensitive to thyroid status. METHODS: A procedure has been developed for the determination of TRH in the thyroid gland, distinct from TRH-like peptides which also react with TRH-antibody. RESULTS: Human thyroid was shown to contain both authentic TRH and TRH-like peptides, a similar pattern was seen in a range of animal thyroids. The concentrations of TRH in non-active goiter thyroids were substantial (41.6-248 pmol.g-1); in contrast the thyroids from hyperthyroid patients contained very little TRH (0.01-2.52 pmol.g-1). CONCLUSION: The physiologic role of TRH in the thyroid is not known but the large difference between the concentrations of this hormone in non-active and hyperactive thyroids suggests that thyroidal TRH may be involved in the regulation of thyroid status.

Identification of the TRH-like peptides pGlu-Glu-Pro amide and pGlu-Phe-Pro amide in rat thyroid: regulation by thyroid status.

Rat thyroid contains thyrotropin-releasing hormone (TRH) and TRH-like peptides which react with TRH antisera. We have identified the TRH-like peptides in the thyroid and examined whether their levels are influenced by thyroid status. The peptides were extracted from the thyroid glands of five hyperthyroid rats and purified by ion-exchange chromatography on SP-Sephadex C25 and reversed-phase high performance liquid chromatography. The principal TRH-immunoreactive component exhibited the same retention on HPLC as synthetic pGlu-Glu-Pro amide and a secondary component corresponded to synthetic pGlu-Phe-Pro amide. In agreement with these assignments the main peptide was shown to be acidic when chromatographed on DEAE-Sephadex A25 and the second peptide neutral. The levels of TRH and TRH-like peptides in the thyroid were investigated in hyper-, hypo- and euthyroid rats. Hyperthyroidism was induced by chronic subcutaneous administration of triiodothyronine (T3) and hypothyroidism was produced by addition of propylthiouracil (PTU) to the drinking water. The amounts of the peptides were determined by radioimmunoassay with a TRH-antiserum, carried out after extraction from the tissues and purification by ion exchange chromatography. The mean concentration of TRH-like peptides in the thyroids of the hyperthyroid rats was 95.5+/-25.5 pmol/g, the mean concentration in the hypothyroid rats was 11.7+/-3.4 pmol/g, and in the euthyroid rats 17.6+/-3.2 pmol/g. The concentrations of TRH were less influenced by thyroid status: the values in hyper-, hypo- and euthyroid rats were 47.5+/-9.4, 42.1+/-6.3, and 17.2+/-1.6 pmol/g respectively. The results show that the levels of the TRH-like peptides in rat thyroid are highly sensitive to thyroid status, suggesting a possible involvement in thyroid regulation.

The thyrotropin-releasing hormone-like peptides pGlu-Phe-Pro amide and pGlu-Glu-Pro amide increase plasma triiodothyronine levels in the mouse; the activity is sensitive to testosterone.

Three naturally occurring peptides, pGlu-Glu-Pro amide, pGlu-Phe-Pro amide and pGlu-Gln-Pro amide, with similar structures to thyrotropin releasing hormone (TRH) have recently been identified but no studies of their in vivo activities have been reported previously. We describe here the ability of pGlu-Phe-Pro amide and pGlu-Glu-Pro amide to influence thyroid status. Subcutaneous administration of these 'TRH-like' peptides in male and female CDI mice led to increased levels of triiodothyronine (T3) and to a lesser extent tetraiodothyronine (T4) in the circulation. pGlu-Phe-Pro amide was more potent than pGlu-Glu-Pro amide; it exhibited a similar potency to pGlu-His-Pro amide (TRH). pGlu-Phe-Pro amide, pGlu-Glu-Pro amide and TRH produced significantly greater effects in the female than in the male. Castration of male mice led to increased activities, with potencies comparable to those seen in the female; in contrast treatment of female mice with testosterone resulted in reduced activities, similar to those observed in the control male. The effects of potassium deprivation on the activities of the TRH-like peptides were also investigated. This diet, which results in decreased testosterone levels in the male, led to increased activities of the TRH-like peptides and TRH, approaching the potencies observed in the female. The results demonstrate that the TRH-like peptides pGlu-Phe-Pro amide and pGlu-Glu-Pro amide which occur naturally in the thyroid gland exhibit biological activity in influencing thyroid status in vivo. The activities are sensitive to testosterone.

Molecular cloning in the marmoset shows that semenogelin is not the precursor of the TRH-like peptide pGlu-Glu-Pro amide.

Two peptides with similar structures to thyrotropin-releasing hormone (TRH), pGlu-Glu-Pro amide and pGlu-Phe-Pro amide, have been identified in human seminal fluid and it has been shown that one of these peptides, pGlu-Glu-Pro amide, has the ability to increase the capacitation of sperm cells, consistent with a role in fertility. In order to select a species in which there is a high degree of expression of the genes that code for 'TRH-like' peptides, we have determined the levels of these peptides in the prostate, pancreas and thyroid of a range of species including rat, rabbit, ox, marmoset, macaque and man. The peptides were extracted from the tissues and purified before determination by RIA with TRH antibody. In addition, trypsin digestion and TRH RIA was used to investigate the presence of N-extended forms. The highest concentrations of TRH-immunoreactive peptides were found in the tissues of the marmoset, Callithrix jacchus. Ion-exchange chromatography demonstrated that marmoset thyroid contained principally authentic TRH, the pancreas contained both TRH and TRH-like peptides while the prostate contained TRH-like peptides alone. Further purification by HPLC showed that the main TRH-immunoreactive peptide in marmoset prostate was pGlu-Glu-Pro amide and a second component was identified as pGlu-Phe-Pro amide. The results indicate that the biosynthesis of these peptides could be studied to advantage in the marmoset. The biosynthetic precursors of the TRH-like peptides have not been identified. To examine whether pGlu-Glu-Pro amide might originate from semenogelin, we determined the sequence of semenogelin in the marmoset. It exhibited a high degree of homology with human semenogelin-I, but in place of the Lys-Gln-Glu-Pro sequence that might give rise to pGlu-Glu-Pro amide, marmoset semenogelin possessed the sequence Ser-Gln-Asp-Gln which cannot serve as a precursor for a TRH-like peptide. Further evidence was obtained by Northern blot analysis of a range of marmoset tissues. The results showed that semenogelin is not present in marmoset prostate. It is concluded that pGlu-Glu-Pro amide originates from a precursor distinct from semenogelin, both in marmoset and in man.

Synthesis and high-performance liquid chromatographic purification of tritiated thyrotrophin-releasing hormone-like peptides.

A simple method is presented for the synthesis and RP-HPLC purification of tritium-labelled thyrotrophin-releasing hormone (TRH)-like tripeptides. These peptides differ from TRH (pGlu-His-Pro-amide) in that they possess a neutral or acidic residue in place of the histidine of TRH. The method involves the preparation of the appropriate dipeptide by a solid-phase peptide synthesis procedure using 9-fluorenylmethoxycarbonyl (Fmoc) protection. Very small amounts of tritiated glutamine are then converted into tritiated pyroglutamic acid, and coupling to the dipeptide is effected using a mixed anhydride derived from Fmoc-phenylalanine and the tritiated pyroglutamic acid. The required labelled product is then separated from unlabelled material by reversed-phase HPLC, as the hydrophobicity of the phenylalanine-containing product ensures that it is strongly retained. The availability of a series of tritium-labelled markers prepared by this method has permitted the unequivocal identification of certain naturally occurring TRH-like peptides.

Isolation and identification of N-terminally extended forms of 5-oxoprolylglutamylprolinamide (Glp-Glu-Pro-NH2), a thyrotropin-releasing-hormone (TRH)-like peptide present in human semen.

N-terminally extended forms of 5-oxoprolylglutamylprolinamide (Glp-Glu-Pro-NH2), a thyrotropin-releasing-hormone(TRH)-like peptide associated with the male reproductive system, were isolated from human semen by gel exclusion on Sephadex G50, ion-exchange chromatography on SP-Sephade C25 and QAE-Sephadex A25, and by HPLC. The peptides were located by trypsin-catalysed release of their C-terminal fragments which were detected by RIA with a TRH-specific antibody. A series of overlapping peptides containing 16, 18, 22 and 25 residues was obtained in homogeneous form and their sequences were determined by automatic Edman degradation. The peptides all terminated in -Lys-Gln-Glu-Pro-NH2 and were found to correspond to sequences occurring between residues 350-374 of semenogelin, a protein present in human semen. In semenogelin, however, the Gln-Glu-Pro sequence is followed by tryptophan and not glycine which is normally essential for formation of the C-terminal amide group. Model experiments with the synthetic peptide Glp-Glu-Pro-Trp showed that under a range of experimental conditions the tetrapeptide did not undergo conversion to Glp-Glu-Pro-NH2. This would indicate that the tripeptide and its extended forms are generated from a precursor that is related to semenogelin but in which Trp375 is replaced by glycine.

The processing of beta-endorphin and alpha-melanotrophin in the pars intermedia of Xenopus laevis is influenced by background adaptation.

beta-Endorphin- and alpha-melanotrophin (alpha-MSH)-related peptides were extracted from the pars intermedia of Xenopus laevis maintained for 2, 4 or 6 weeks on a white background and for the same periods on a black background. The peptides were resolved under dissociating conditions by gel exclusion chromatography on Sephadex G-50 and they were detected by radioimmunoassay with antibodies to beta-endorphin, alpha,N-acetyl beta-endorphin and alpha-MSH. The beta-endorphin-related peptides separated into two fractions of different molecular size. Further purification of the peptides in each fraction was by ion exchange chromatography on SP-Sephadex C-25 and by high-pressure liquid chromatography. The alpha-MSH-related peptides were resolved by gel exclusion and ion exchange chromatography. The purified beta-endorphin- and alpha-MSH-immunoreactive peptides were identified by comparison of their chromatographic properties with the corresponding peptides from porcine pituitary or by comparison with synthetic peptides. The major form of beta-endorphin in the pars intermedia of the frog adapted to a white background was identified as alpha,N-acetyl beta-endorphin (1-8); it was accompanied by a small quantity of acetylated peptides with molecular size similar to beta-endorphin. In contrast, the pars intermedia of the frogs adapted to a black background contained approximately equal amounts of alpha,N-acetyl beta-endorphin (1-8) and the larger forms of beta-endorphin. The higher molecular weight forms were identified as the alpha,N-acetyl derivatives of beta-endorphin (1-26), (1-27) and (1-31); however after 6 weeks of white adaptation the sole remaining peptide in this group was the 26-residue peptide. An additional beta-endorphin immunoreactive peptide, provisionally identified as beta-endorphin (10-26), was present in both black- and white-adapted animals; the amounts of this peptide increased during white adaptation. Major differences in the processing of alpha-MSH were also observed. In the frogs adapted to a black background des-acetyl alpha-MSH greatly predominated over the acetyl form whereas after 6- weeks adaptation to a white background the acetylated peptide proved to be the principal component. The results demonstrate that the proteolytic processing of beta-endorphin and the acetylation of alpha-MSH in Xenopus laevis are influenced by background adaptation. The formation of beta-endorphin (1-8) appears to reflect the action of an endopeptidase that acts at the single arginine residue present at position 9.(ABSTRACT TRUNCATED AT 400 WORDS)

Isolation and identification of two neutral thyrotropin releasing hormone-like peptides, pyroglutamylphenylalanineproline amide and pyroglutamylglutamineproline amide, from human seminal fluid.

Two tripeptide amides with stuctures similar to thyrotropin releasing hormone were isolated from human seminal fluid and their amino acid sequences determined. The peptides were purified by gel exclusion from Sephadex G50 and were detected by radioimmunoassay with thyrotropin releasing hormone antibody; in addition, N-terminally extended forms were demonstrated by radioimmunoassay after trypsin digestion. Further purification of the tripeptides was by chromatography on SP-Sephadex C25 and by high performance liquid chromatography on C18 Microbondapak using an HCl/acetonitrile gradient. After exclusion from mini-columns of SP-Sephadex C25 and DEAE-Sephadex A25, two neutral peptides were obtained in homogeneous form by high performance liquid chromatography with an HCl/methanol gradient. Amino acid analysis gave the following compositions: Glu, 0.74, Phe, 1.0, Pro, 1.0; and Glu, 1.72, Pro, 1.0. Both peptides possessed a blocked N terminus, but after opening the pyroglutamyl ring the sequences Glu-Phe-Pro and Glu-Glx-Pro were demonstrated. The chromatographic properties of the endogenous peptides were identical to the properties of the corresponding synthetic peptides. The structure of pGlu-Phe-Pro (where p-indicates pyro-) amide was confirmed by fast atom bombardment mass spectrometry. The presence in human semen of three structurally related peptides, pGlu-Phe-Pro amide, pGlu-Gln-Pro amide, and the previously reported pGlu-Glu-Pro amide (Cockle, S. M., Aitken, A., Beg, F., and Smyth, D. G. (1989) J. Biol. Chem. 264, 7788-7791), suggests that this series of peptides may have evolved to fulfil complementary biological roles.

The thyrotrophin-releasing hormone (TRH)-like peptides in rat prostate are not formed by expression of the TRH gene but are suppressed by thyroid hormone.

Thyrotrophin-releasing hormone (TRH)-immunoreactive peptides were extracted from rat prostate and divided into two groups by mini-column cation exchange chromatography. The amounts of the peptides in each group were determined by radioimmunoassay with a TRH antiserum. The unretained peptides which lacked a basic group and the retained peptides which possessed a basic group were further purified by high-performance liquid chromatography. The unretained fraction was found to contain a series of TRH-immunoreactive peptides, one of which corresponded chromatographically to synthetic pGlu-Glu-Pro amide and another to pGlu-Phe-Pro amide. None of the TRH-immunoreactive peptides in either fraction exhibited the chromatographic behaviour of TRH. Additional evidence for the absence of TRH gene expression in the prostate was obtained by Northern blot analysis and by application of polymerase chain reaction amplification, which failed to reveal TRH mRNA. Furthermore the preproTRH-derived peptide, preproTRH(53-74), could not be detected by radioimmunoassay. The influence of thyroid status was investigated on the levels of the TRH-like peptides in the prostate. Adult rats were treated chronically with thyroxine (T4) or propylthiouracil (PTU) and the concentrations of the TRH-immunoreactive peptides were determined by chromatography and radioimmunoassay. Treatment with T4 caused the levels of the neutral and acidic TRH-like peptides to fall to approximately one-third of the levels in the controls. No significant difference from the controls was seen in the concentrations of the peptides in the prostates of rats rendered hypothyroid by administration of PTU. The results demonstrate that rat prostate contains TRH-immunoreactive peptides which are not derived from the TRH gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Peptide amidation.

Many hormones, neurotransmitters and growth factors are peptides that carry an amide group at their carboxyl terminus which is essential for their biological activity. The amide is formed by hydroxylation of an additional glycine residue present in the biosynthetic precursor and the hydroxyglycine derivative dissociates to form the peptide amide and glyoxylic acid. Recent discoveries have shown that two enzymes are involved that act sequentially.

Purification of a cysteine endopeptidase which is secreted with bioactive peptides from the epidermal glands of Xenopus laevis.

The purification is reported of an endopeptidase, XSCEP1 (Xenopus skin cysteine endopeptidase), present in skin secretions of Xenopus. The procedure involved an initial concentration of the enzyme by batchwise anion-exchange chromatography and ammonium sulphate precipitation. The proteolytic activity, determined with Z-Phe-Arg-Amc (Z, benzyloxycarbonyl; Amc, 7-amidomethylcoumarin) as substrate, was fractionated by gradient ion-exchange chromatography, yielding a major component which was purified to homogeneity by chromatography on an organomercury-agarose column. SDS/PAGE demonstrated the presence of a single protein with a molecular mass of 27 kDa. The purified enzyme, which possessed a pH optimum of 5.5, exhibited the properties of a cysteine endopeptidase; it was activated by dithiothreitol and EDTA and inhibited by the mechanism-based inhibitor trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane. XSCEP1 exhibited a marked preference for substrates with a hydrophobic residue in the P1 position and arginine in the P2 position as opposed to a substrate with arginine residues in both positions. The enzyme was also able to cleave a Val-Arg-Gly sequence in a model substrate, reflecting cleavages undergone by a number of peptides present in Xenopus skin. The results point to a functional role for XSCEP1 as a putative processing enzyme.

Distribution of pyroglutamylpeptide amides related to thyrotrophin-releasing hormone in the central nervous system and periphery of the rat.

A rapid and sensitive chromatographic method is presented for determining TRH and TRH-like peptides in small quantities of tissues and fluids. The procedure was applied to screen a comprehensive range of tissues from the central nervous system (CNS) and periphery of the rat. When hydrochloric acid (100 mmol/l) was used for extraction, the TRH immunoreactivity obtained was identified solely with the known tripeptide hormone. In contrast, when weakly acidic conditions were used for extraction, neutral or acidic TRH-like peptides which lacked the histidine residue at position 2 of the hormone, but reacted with TRH antibodies, were shown to be present in addition to TRH. The TRH-like peptides in the brain were located principally in the hippocampus, brain stem and dorsal colliculi. In the periphery, TRH-like peptides were shown to be present in the male reproductive system and certain endocrine tissues. In addition, the presence of TRH-like peptides was confirmed in portal blood. The results indicate that peptides with a neutral or acidic TRH-like sequence at their C-terminus are widely distributed in the CNS and periphery of the rat. These peptides appear to occur mainly or exclusively in precursor forms from which TRH immunoreactivity can be released during extraction under weakly acidic conditions.

4-Phenyl-3-butenoic acid, an in vivo inhibitor of peptidylglycine hydroxylase (peptide amidating enzyme).

The ability of a series of non-peptide carboxylic acids to act as substrates or inhibitors of the peptide-amidating enzyme (peptidyl-glycine hydroxylase) was assessed by determining their ability to reduce the rate of enzymic conversion of D-tyrosyl-valyl-glycine or D-tyrosyl-phenylalanyl-glycine to the corresponding dipeptide amide. The inclusion of a phenyl substituent in a position distal to the carboxyl group promoted the inhibitory action. The inhibition was found to be irreversible when an olefinic double bond, alpha or beta to the carboxyl group, was present in the molecule; the inhibition appeared to be associated with a covalent interaction between the amidating enzyme and the inhibitor. With 4-phenyl-3-butenoic acid the inhibitory properties were manifest only in the presence of cofactors of the enzyme. When 4-phenyl-3-[2-14C]butenoic acid was used, the radioactivity was shown to be incorporated into protein that co-chromatographed with active enzyme. Incubation of rat thyroid carcinoma CA77 cells in the presence of 4-phenyl-3-butenoic acid led to a decrease in the levels of intracellular amidating activity and of thyrotropin-releasing hormone, an amidated peptide produced by these cells. The inhibitory effects reached a maximum at approximately 15 h after which the enzyme levels returned to the control values even though the concentration of 4-phenyl-3-butenoic acid in the cells remained unchanged. The results indicate that a mechanism exists in these cells for regulation of amidating activity.

Endopeptidases and prohormone processing.

Peptide hormones and peptide transmitters are generated from polypeptide precursors by specific cleavage reactions which take place principally at sites formed by single or paired basic residues. Not all the possible cleavage sites are utilised, however, and the degree of processing of many propeptides has been found to vary according to the tissue of origin. The restricted nature of processing reactions could point to the existence of a series of enzymes with stringent specificities, recognising regions of structure in addition to the single or paired basic residues. Alternatively the action of processing enzymes may be directed by conformation of the pro-peptide which could focus the action of a protease onto or away from a particular site. In addition certain post-translational modifications such as glycosylation or phosphorylation may influence the accessibility of a site to the approach of a processing enzyme. In this review we describe recent advances that have been made in the characteristization of proteolytic processing enzymes, we examine the relevance of the various factors that could account for restricted processing and discuss new approaches that may lead to better understanding of the mechanisms involved.

Catalysis of slow C-terminal processing reactions by carboxypeptidase H.

A hypothesis was examined that carboxypeptidase H (CpAse H), which is known to catalyse the release of lysine and arginine from the C-terminus of peptides, can also release histidine, tyrosine, and phenylalanine. Synthetic peptides terminating in -His-Lys or -Tyr-Lys were used as model substrates for the enzyme and amino acid analysis was employed to detect release of the terminal amino acids. With N-acetyl-beta-Ala-Asn-Ala-His-Lys and N-acetyl-beta-Ala-Asn-Ala-Tyr-Lys, which correspond to intermediates in the processing of porcine and human beta-endorphin, lysine was removed rapidly and quantitatively but no release of histidine or tyrosine could be detected. To allow more sensitive analysis, radiolabelled substrates were employed and the amounts of the products formed on incubation with CpAse H were determined after separation by ion-exchange chromatography. With 125I-D-Tyr-Ala-His-Lys-Lys as substrate at pH 5.7, very small amounts of D-Tyr-Ala were released; the main product was D-Tyr-Ala-His. At pH 5.0 the release of histidine from 125I-D-Tyr-Ala-His took place 6,000 times more slowly than the release of lysine from 125I-D-Tyr-Ala-Lys. When the tripeptides were incubated at pH 5 with porcine pituitary secretory granules, the lysine was released rapidly but no release of histidine could be detected. The results demonstrate that CpAse H catalyses the release of C-terminal histidine with great difficulty.(ABSTRACT TRUNCATED AT 250 WORDS)