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Biomedical subjects

D G Smyth

Publications and source records attributed to D G Smyth.

At least 19 recordsLinked to original sources
PubMed1992

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.

Amino Acid Sequence↗
PubMed1992

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)

Animals↗
PubMed1992

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)

Acetylation↗
PubMed1991

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.

Amino Acid Sequence↗
PubMed1991

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.

Amidine-Lyases↗
PubMed1990

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.

Amino Acid Sequence↗
PubMed1990

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.

Amino Acid Sequence↗
PubMed1990

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.

Animals↗
PubMed1989

The TRH-related peptide pyroglutamyglutamylprolinamide is present in human semen.

We have recently identified a novel peptide in the rabbit prostate complex which cross-reacts with an antibody to thyrotrophin-releasing hormone (TRH) and has the structure pGlu-Glu-ProNH2. In the present study, high concentrations of a TRH-related tripeptide and also a polypeptide (10-12 kDa) containing a TRH-immunoreactive peptide at its C-terminus were detected in human semen. The low molecular mass TRH-like peptide and the immunoreactive fragment from the polypeptide were isolated from human semen and shown to have identical structures. Amino acid analysis suggested compositions Glx2, Pro1, and after mild acid hydrolysis, the same sequence, Glu-Glu-Pro, was established for the two peptides. Fast atom bombardment (FAB) mass spectrometry yielded a pseudomolecular ion (M + H)+ of 355.38 which was identical to that of the synthetic peptide pGlu-Glu-ProNH2. The data demonstrate that human semen contains the TRH-like peptide pyroglutamylglutamylprolinamide and also a polypeptide terminating in the sequence Gln-Glu-ProNH2.

Adult↗
PubMed1989

A novel peptide, pyroglutamylglutamylproline amide, in the rabbit prostate complex, structurally related to thyrotrophin-releasing hormone.

A novel peptide which cross-reacts with an antibody to thyrotrophin-releasing hormone has been isolated and characterized from the rabbit prostate complex. The peptide exhibited an amino acid composition of Glx1.7, Pro1.0, and automatic gas phase sequence analysis after mild acid hydrolysis established the sequence Glu-Glu-Pro. Fast atom bombardment mass spectrometry gave a pseudomolecular ion (M + H)+ of 355.2 confirming that the prostate peptide has the structure of pGlu-Glu-Pro-NH2. This peptide differs from authentic thyrotrophin-releasing hormone by the substitution of glutamic acid for histidine at position 2.

Amino Acid Sequence↗
PubMed1989

Continuous infusion of propofol in dystrophia myotonica.

The use of intravenous infusions of propofol and atracurium in a 41-year-old woman, weighing 64 kg, with dystrophia myotonica during major oral surgery is described. Propofol and atracurium, in total doses of 2488 mg and 75 mg respectively, were used during the four-hour procedure. There were no intraoperative problems and operating conditions were excellent. Emergence was rapid and there were no postoperative complications. This technique offers a safe alternative to inhalational anaesthesia for patients with dystrophia myotonica.

Adult↗
PubMed1989

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)

Amino Acid Sequence↗
PubMed1989

Thyrotrophin-releasing hormone-related polypeptides in rabbit prostate and semen are different from those in rabbit hypothalamus.

TRH-related peptides were extracted from the hypothalamus and prostate gland of the rabbit. The peptides were fractionated by gel exclusion chromatography and located by trypsin digestion and radioimmunoassay with antibodies to TRH amide and TRH-Gly Lys. In the hypothalamus TRH-related peptides containing approximately 16 and 30 residues were observed: in these peptides the extensions to the TRH sequence were exclusively in the C-terminal direction. In addition, the three-residue form of TRH was also present. In the prostate complex, the predominant TRH-related peptide contained approximately 50 residues and the extension to the TRH tripeptide was on the N-terminal side; a three-residue form of immunoreactive TRH was also demonstrated. The same pattern of TRH-related peptides was shown to be present in rabbit semen. The results reveal the existence of a novel TRH-related polypeptide in the prostate and semen which does not occur in the hypothalamus. This peptide appears to undergo secretion.

Animals↗
PubMed1988

Peptide amidation: evidence for multiple molecular forms of the amidating enzyme.

Amidating enzyme extracted from porcine pituitary was separated into glycosylated and non-glycosylated forms by fractionation on a column of Concanavalin-A Sepharose. The molecular weights of the species present were assessed by HPLC gel exclusion chromatography, which demonstrated that both the glycosylated and the non-glycosylated forms of the enzyme comprise multiple components. The apparent molecular weights of the non-glycosylated forms ranged from approximately 35 kDa to 100 kDa; the glycosylated enzyme contained species with molecular weights ranging from 65 kDa to 135 kDa. Similar proportions of glycosylated to non-glycosylated enzyme (approximately 1:4) were found in the anterior and posterior regions of the pituitary; higher proportions (approximately 1:1) were observed in the thyroid, adrenals and pancreas. The glycosylated forms of the amidating enzyme were shown to exhibit the same mandatory requirement for copper as the non-glycosylated forms, and no differences were seen in respect of their stimulation by dopamine or their pH optima. Both forms catalysed the hydroxylation of glyoxylic acid phenylhydrazone, indicating a common mechanism of action. By these criteria, glycosylation does not affect the activity of the amidating enzyme.

Animals↗
PubMed1988

Substrate recognition by UDP-N-acetyl-alpha-D-galactosamine: polypeptide n-acetyl-alpha-D-galactosaminyltransferase. Effects of chain length and disulphide bonding of synthetic peptide substrates.

A synthetic peptide AcTPPP, based on a threonine-containing sequence present in bovine myelin basic protein, is a potent acceptor of glycosyl transfer from UDP-N-acetylgalactosamine catalyzed by extracts of baby hamster kidney (BHK) cells or rabbit lymph node tissue. In contrast, the disulphide-linked peptide (AcTCPPP)2, based on a glycosylated sequence present in the hinge region of rabbit immunoglobulin G, is not an acceptor and inhibits glycosylation of AcTPPP. Extension of the cystine-containing peptide at the N-terminus produced weak acceptors but strong acceptors resulted when the cystine residue was reduced to form monomeric peptides. The acceptor specificity of the N-acetylgalactosaminyl-transferase activity of BHK cells is very similar to that of rabbit lymph node tissue. The results indicated that tissues actively secreting immunoglobulin do not contain a transferase activity adapted specifically for glycosylation of sequences containing cystine residues, and suggested that addition of an N-acetylgalactosamine to a threonine residue in the hinge region of rabbit immunoglobulin takes place during biosynthesis prior to the formation of the inter-chain disulphide bridge of fully assembled immunoglobulins.

Amino Acid Sequence↗