Biomedical subjects

D G Smyth

Publications and source records attributed to D G Smyth.

At least 37 records · Page 2Linked to original sources

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↗

PubMed1988

Characterization of a proteolytic enzyme in the skin secretions of Xenopus laevis.

Enzymes present in the skin secretions of Xenopus laevis were fractionated by ion exchange chromatography. One of the proteases obtained was found to catalyse cleavage on the COOH-side of peptide sequences containing consecutive hydrophobic and basic residues. Evidence is presented that the enzyme is a cysteine protease with an optimum pH of 5.0 to 6.0. The characteristic specificity of this enzyme suggests that it may fulfil a role in propeptide processing.

Animals↗

PubMed1988

The glycopeptide moiety of vasopressin-neurophysin precursor is neurohypophysial prolactin releasing factor.

All of the classically-described hypothalamic, hypophysiotropic factors that regulate anterior pituitary hormone secretion have now been isolated and identified except for prolactin releasing factor. We report here that the 39-amino acid glycopeptide comprising the carboxyterminus of the neurohypophysial vasopressin-neurophysin precursor stimulates prolactin release from cultured pituitary cells as potently as does thyrotropin releasing hormone but has no effect on the secretion of other pituitary hormones. Furthermore, antisera to the glycopeptide administered to lactating rats attenuated suckling-induced prolactin secretion. Thus, this glycopeptide appears to be the neurohypophysial prolactin releasing factor.

Animals↗

PubMed1988

Processing reactions in the later stages of hormone activation.

Three tiers of processing have been investigated in the reactions that transform prohormones into their mature end products. Evidence is presented that the proteolytic reactions that convert lipotropin into shortened forms of beta-endorphin take place in individually distinct stages. After these cleavages have occurred, the removal of basic residues by carboxypeptidase H and amidation of the products are effected by independent reactions which do not synergise. Experiments are also described which show that the amidating enzyme can accept certain imino acids as substrates and utilises a mechanism that involves hydroxylation; it is implicit that peptide amidation proceeds by a similar mechanism. These results point to a general concept that pro-hormone processing involves consecutive reactions which take place in a predetermined order.

Animals↗

PubMed1988

Sequential formation of beta-endorphin-related peptides in porcine pituitary.

Lipotropin and peptides related to beta-endorphin were extracted from the anterior pituitary and the pars intermedia of porcine pituitary and were resolved by gel exclusion and ion exchange chromatography. Possible heterogeneity in the structure of the lipotropin was investigated by identifying the C-terminal fragment released by limited proteolysis with trypsin; the cleavage was restricted to the carboxyl group of arginine residues by employing citraconylation to protect the epsilon-NH2 groups of lysine. The lipotropin obtained from both regions of the pituitary gave rise to the same C-terminal peptide which contained the 31-residue sequence of beta-endorphin; none of the 26- and 27-residue forms was detected. In contrast, the beta-endorphin-related peptides that were isolated directly from the pars intermedia exhibited a high degree of C-terminal proteolysis: they were present principally as the 26- and 27-residue peptides. The results demonstrate that lipotropin differs from beta-endorphin in that it occurs exclusively in the form that contains the full C-terminal sequence. It is concluded that during biosynthesis lipotropin undergoes conversion to beta-endorphin before proteolysis takes place at the C-terminus. The processing reactions that convert lipotropin to beta-endorphin 1-31 and beta-endorphin 1-31 to beta-endorphin 1-27 are thus ordered and not competitive. The results also indicate that glycylglutamine, the bioactive C-terminal dipeptide of lipotropin, is formed from beta-endorphin and not from lipotropin.

Animals↗

PubMed1988

Biosynthesis of peptide neurotransmitters: studies on the formation of peptide amides.

A high proportion of peptide transmitters and peptide hormones terminate their peptide chain in a C-terminal amide group which is essential for their biological activity. The specificity of an enzyme that catalyses the formation of the amide was investigated with the aid of synthetic peptide substrates. With peptides containing l-amino acids the enzyme exhibited an essential requirement for glycine in the C-terminal position; amidation did not take place with peptides that had leucine, alanine, glutamic acid, lysine or N-methylglycine at the C-terminus and a peptide extended by the attachment of lysine to the C-terminal glycine did not act as a substrate. Amidation did occur with a peptide containing C-terminal D-alanine but no reaction was detected with peptides having C-terminal, D-serine or D-leucine. In tripeptides with a neutral amino acid in the penultimate position, amidation, took place readily but the reaction was slower when this position was occupied by an acidic or a basic residue. A series of overlapping peptides with C-terminal glycine, based on partial sequences of calcitonin, underwent amidation at similar rates, indicating that the amidating enzyme recognizes only a limited sequence at the C-terminus of its substrates. The results provide evidence that the amidating enzyme has a highly compact substrate binding site.

Amino Acid Sequence↗

PubMed1987

Enzyme-catalysed peptide amidation. Isolation of a stable intermediate formed by reaction of the amidating enzyme with an imino acid.

A series of hydrazones and semicarbazones of glyoxylic acid were shown to have a potent inhibitory effect on the enzyme-catalysed conversion of D-Tyr-Val-Gly to D-Tyr-Val-NH2. Among the derivatives tested, the inhibitory activity was increased by the presence of hydrophobic substituents and decreased by polar substituents. The inhibition produced by glyoxylic acid phenylhydrazone was shown to be competitive. No inhibition was obtained with pyruvic acid phenylhydrazone, which possesses a methyl group in place of the alpha-H of glyoxylic acid phenylhydrazone. The inhibitory potencies of these non-peptide substances are in accord with the specificity exhibited by the amidating enzyme in its reaction with peptide substrates. The inhibition produced by the glyoxylic acid derivatives was shown to be due to their ability to act as substrates for the peptide-amidating enzyme. The product formed from [14C]glyoxylic acid phenylhydrazone was identified as oxalic acid phenylhydrazide by co-chromatography in three chromatographic systems. The results demonstrate that the enzyme-catalysed oxidation of glyoxylic acid phenylhydrazone takes place by a mechanism involving hydroxylation. It is implicit that peptide amidation catalysed by the same enzyme proceeds by a similar mechanism.

Amidohydrolases↗

PubMed1987

Sequential processing reactions in the formation of hormone amides.

The substrate specificity of an enzyme with amidating activity, present in porcine pituitary, was investigated by examining its ability to convert the synthetic peptides D-Tyr-Val-Gly and D-Tyr-Val-Gly-Lys-Arg to the dipeptide amide D-Tyr-Val-CONH2. The purified enzyme catalysed the amidation reaction with the tripeptide but did not accept the pentapeptide as a substrate. With the mixture of enzymes present in a membrane fraction from porcine pituitary or the enzymes in a secretory granule fraction, both the tripeptide and pentapeptide substrates gave rise to D-Tyr-Val amide; the formation of dipeptide amide from the pentapeptide, however, involved a latency period after which amidation occurred at a similar rate with the two substrates. Evidence was obtained that arginine and lysine were released from the C terminus of the pentapeptide before amidation took place since the rate of formation of dipeptide amide was reduced at pH values that were compatible with amidation but unfavourable to the action of carboxypeptidase H. In addition formation of the dipeptide amide from the pentapeptide was blocked by guanidinoethylmercaptosuccinic acid and glycylarginine, which are inhibitors of carboxypeptidase enzymes. The experiments demonstrate that removal of basic residues from the C terminus of a peptide and amidation at C-terminal glycine are reactions that take place consecutively. These prohormone-processing reactions, which are intrinsic to the formation of hormone amides, did not synergise.

Amidohydrolases↗

PubMed1987

Specific processing of the thyrotropin-releasing prohormone in rat brain and spinal cord.

Thyrotropin-releasing hormone (TRH) and TRH extended peptides were extracted from rat hypothalamus and spinal cord and resolved by gel exclusion chromatography under dissociating conditions. Peptides related to TRH were detected by trypsin digestion and radioimmunoassay with an antibody to TRH or an antibody raised against the pentapeptide Glp-His-Pro-Gly-Lys. In addition to the tripeptide hormone a series of C-terminally extended forms of TRH was shown to occur in both tissues; no N-terminally extended peptides were detected. The structure of the TRH-related peptides was confirmed by chromatographic identification of the N-terminal pentapeptide sequence released by trypsin. The TRH extended peptides, which accounted for 15-20% of the total TRH, were present in three groups of different molecular size corresponding to predicted fragments of the TRH prohormone. One of the peptides in the spinal cord was identified by chromatographic comparison with a synthetic 16-residue peptide representing residues 154-169 of the prohormone. In the spinal cord the TRH extended peptides differed in their relative concentrations from the corresponding peptides in the hypothalamus, possibly reflecting differences in processing. The finding of extended forms of TRH in which the extension occurs only on the C-terminal side of the hormone sequence shows that the prohormone undergoes highly specific processing.

Animals↗

PubMed1987

Biosynthesis of the C-terminal amide in peptide hormones.

Recent developments in the study of peptide amidation are reviewed. The main areas covered are assay procedures, purification of amidating enzymes, co-factors and regulation, mechanism and specificity of the amidating reaction, and multiple forms of the amidating enzyme and glycosylation. Discussion is presented on aspects that are poorly understood and new areas open to investigation are indicated.

Amides↗

PubMed1986

Beta-endorphin is present in active and inactive forms in rat gastric antrum.

Lipotropin, beta-endorphin and a series of peptides related to beta-endorphin were extracted from rat antrum and resolved by gel filtration, ion exchange chromatography and high pressure liquid chromatography; the concentrations of the peptides were determined by radioimmunoassay. The major peptide with beta-endorphin immunoreactivity present in the antrum was lipotropin but it was accompanied by substantial quantities of beta-endorphin in its biologically active form; in addition there were minor quantities of a number of inactive beta-endorphin related peptides. The experiments demonstrate that in rat antrum gastrin can be accompanied by both active and inactive forms of beta-endorphin. The implications of post-translational processing mechanisms common to gastrin and beta-endorphin are discussed.

Animals↗

PubMed1986

Detection of TRH extended peptides in rat hypothalamus using an antibody raised to pGluHisProGlyLys.

An antibody was raised to the synthetic pentapeptide pGluHisProGlyLys which, in radioimmunoassay (RIA), could detect the pentapeptide at a level of 10 fmole per tube and exhibited less than 0.5 per cent cross reactivity with a series of related peptides. The RIA was used to demonstrate the presence of C-terminally extended forms of thyrotropin releasing hormone (TRH) in rat hypothalamus. After extraction, the endogenous peptides were resolved by gel exclusion chromatography and TRH-extended peptides were revealed by trypsin digestion to release the pentapeptide. The TRH extended peptides occurred in substantial quantity, approximately 11 pmoles/g, indicating that only partial processing of the gene duplicated prohormone takes place.

Animals↗