Thyrotropin-releasing hormone-degrading enzyme in human serum is classified as type II of pyroglutamyl aminopeptidase: influence of thyroid status

The characteristics of thyrotropin-releasing hormone (TRH)-degrading enzyme in human serum were studied. Serum was incubated in 0.1 M phosphate buffer containing [proline-3H]TRH at 37 degrees C. A thin layer chromatography analysis of TRH degradation did not show any radioactive peak located in an acid TRH position, but apparent radioactive peaks corresponding to His-Pro and His-ProNH2 occurred in the presence of p-hydroxymercuriphenyl sulfonic acid, an inhibitor of proline dipeptidase. With ion exchange paper chromatography, the formation of 3H-labeled His-Pro and His-ProNH2 was estimated as an end point in the measurement of pyroglutamyl aminopeptidase (pGlu-peptidase) activity. An assay using p-hydroxymercuriphenyl sulfonic acid was developed to sensitively quantitate the pGlu-peptidase. Neither bacitracin nor p-chloromercuribenzoic acid increased the activity of pGlu-peptidase. The addition of EDTA, dithiothreitol, and o-phenanthroline significantly inhibited pGlu-peptidase activity, but neither iodoacetamide nor ethylmaleimide altered its activity. The pGlu-peptidase had a stereotypic specificity for the tripeptide, pGlu-His-ProNH2 of TRH, and its Km was 44.9 microM. The pGlu-peptidase activity was not changed by either hyper- or hypothyroidism. The present data indicate that a TRH-degrading enzyme in human serum possesses a nature identical to type II of pGlu-peptidase which is not altered by thyroid status.     

Mechanisms of brain inactivation of centrally-acting thyrotrophin-releasing hormone (TRH) analogues: a high-performance liquid chromatography study

High-performance liquid chromatography (HPLC) was used to investigate the degradation in vitro of several centrally-acting analogues of thyrotrophin-releasing hormone (TRH) by two subcellular fractions prepared from different areas of rat brain. Of the seven analogues studied, RX77368 (pGlu-His-(3,3'-dimethyl)-ProNH2) was the most stable analogue, showing only a small amount of degradation by the particulate fraction containing a pyroglutamyl aminopeptidase, whereas the other analogues (RX74355, CG3509, CG3703, [3MeHis]TRH, PGHPA and MK771) showed varying degrees of resistance to degradation by this enzyme and the proline endopeptidase in the soluble fraction. However, TRH was rapidly inactivated to its deamidated form, TRH-OH and the histidyl-proline diketopiperazine by both fractions. The relative stability of these TRH analogues to enzyme action may provide some explanation for their enhanced biological activity in vivo.     

Thyrotrophin-releasing hormone inactivation by human postmortem brain

The mechanisms of inactivation of thyrotrophin-releasing hormone (TRH) by peptidases in several areas of normal human postmortem brain have been investigated by radioimmunoassay and high-performance liquid chromatography. Of the several brain regions studied, the cerebral cortex (Brodman's area, BA10) had the highest TRH-degrading activity in both subcellular fractions. Deamidated-TRH (TRH-OH) was the only product formed by the soluble fraction whereas the histidyl-proline diketopiperazine, cyclo(His-Pro), and a small amount of TRH-OH were formed by the particulate fraction. Several centrally acting TRH analogues showed varying degrees of resistance to degradation by the peptidases in the two fractions, the most stable analogue being RX77368 (pGlu-His-3,3'-dimethyl(ProNH2]. Areas of human postmortem brain appear to contain two of the enzymes capable of degrading TRH, a proline endopeptidase forming TRH-OH and a pyroglutamyl aminopeptidase forming cyclo(His-Pro). The use of the assay procedures in further studies on the inactivation of TRH by peptidases from brain areas of patients with neurological disorders may provide complementary information on the dynamics of TRH in these disorders. The stability of the centrally acting TRH analogues may prove useful in examining their therapeutic potential.     

A cDNA from brain of Xenopus laevis coding for a new precursor of thyrotropin-releasing hormone.

Thyrotropin-releasing hormone (TRH) is found in large amounts in the skin of Xenopus laevis. In this tissue, 3 TRH precursor mRNAs can be detected of which the 2 more expressed encode almost identical proteins. However, Northern blot analysis of TRH precursor mRNAs in the brain of X. laevis revealed the existence of a new mRNA of about 1200 nucleotides which was present along with the larger TRH precursor mRNA identified in the skin. A cloned cDNA of a TRH precursor, corresponding in size to this new mRNA, was isolated and sequenced from a Xenopus brain lambda gt11 library. It encodes a precursor polypeptide which also contains 7 copies of TRH. However, at the amino acid level it differs by about 16% from the corresponding prepro-TRHs from skin. We have also attempted to characterize the gene encoding this prepro-TRH from Xenopus brain. Only the first and part of the second exon could be detected which are separated by an intron containing more than 8000 base pairs. Interestingly, the 5'-flanking region of this gene does not contain the characteristic promoter elements of the mammalian TRH genes suggesting marked differences in the regulation of their expression.     

Post-proline dipeptidyl-aminopeptidase from synaptosomal membranes of guinea-pig brain. A possible role for this activity in the hydrolysis of His-ProNH2, arising from the action of synaptosomal membrane pyroglutamate aminopeptidase on thyroliberin

In this paper we report that while 55% of the total post-proline dipeptidyl-aminopeptidase activity in guinea-pig brain is associated with the soluble fraction of the cells, the remaining activity is widely distributed throughout the particulate fractions. A significant portion of this particulate activity is, however, associated with a synaptosomal membrane fraction. The specific activity of this enzyme rose as the synaptosomal membrane fraction was prepared from a synaptosomal fraction and had previously risen at the synaptosomal fraction was prepared from a postmitochondrial pellet. The synaptosomal membrane post-proline dipeptidyl-aminopeptidase was released from the membrane by treatment with Triton X-100 and partially purified by chromatography on Sephadex G-200. By contrast with the soluble enzyme the partially purified solubilised synaptosomal membrane post-proline dipeptidyl-aminopeptidase was not inhibited by 1.0 mM p-chloromercuribenzoate, 1.0 mM N-ethylmaleimide or 0.5 mM puromycin but was inhibited by 0.5 mM bacitracin. The partially purified solubilised enzyme was capable of releasing His-Pro from His-Pro-Val, His-Pro-Leu, His-Pro-Phe and His-Pro-Tyr and of releasing Gly-Pro from Gly-Pro-Ala but could not release Arg-Pro from Arg-Pro-Pro or from Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg (bradykinin). It was also unable to release Pro-Pro from Pro-Pro-Gly or Glp-Pro from Glp-Pro-Ser-Lys-Asp-Ala-Phe-Ile-Gly-Leu-MetNH2 (eledoisin). Using [Pro-3H]thyroliberin we show that the membrane-bound enzyme converts His-ProNH2, produced by the action of the synaptosomal membrane pyroglutamate aminopeptidase, to His-Pro thus competing with the spontaneous cyclisation of His-ProNH2 to His-Pro diketopiperazine. Purified preparations of synaptosomal membrane pyroglutamate aminopeptidase were used to generate His-ProNH2, which could then be converted to His-Pro by the presence of the partially purified synaptosomal membrane post-proline dipeptidyl-aminopeptidase. This preparation was free of contaminating post-proline cleaving endopeptidase, carboxypeptidase P, aminopeptidase P, prolyl carboxypeptidase or proline dipeptidase.     

The chemistry and biology of thymosin. I. Isolation, characterization, and biological activities of thymosin alpha1 and polypeptide beta1 from calf thymus.

A partially purified extract from thymus tissue termed thymosin Fraction 5 has been shown to reconstitute immunological deficiencies resulting from the lack of thymic function in several animal models, as well as humans with primary and secondary immunodeficiency diseases. Thymosin Fraction 5 consists of a family of polypeptides with molecular weights ranging from 1,000 to 15,000. Several of these polypeptides contribute individually to the biological activity of the parent compound. Two polypeptide components of thymosin Fraction 5, termed thymosin alpha1 and polypeptide beta1, have been characterized chemically and biologically. Thymosin alpha1 is a highly acidic molecule composed of 28 amino acid residues. This polypeptide has potent biological activity and has been found to be 10 to 1,000 times as active as thymosin Fraction 5 in one in vivo and several in vitro bioassay systems designed to measure differentiation and function of thymus-dependent lymphocytes (T cells). Polypeptide beta1, in contrast, is inactive in our bioassay systems, suggesting that it is not involved in thymic hormone action. Sequence analysis and homology studies have indicated that polypeptide beta1, although present in Fraction 5, does not contribute to the biological activity of thymosin Fraction 5.     

Degradation and biological inactivation of thyrotropin releasing hormone (TRH): regulation of the membrane-bound TRH-degrading enzyme from rat anterior pituitary by estrogens and thyroid hormones

Thyrotropin releasing hormone (TRH, pyroGlu-His-Pro-NH2) is important in the regulation of adenohypophyseal hormone secretion and also serves important functions in extrahypothalamic brain areas, indicating that it is involved in neurotransmission and other forms of cellular communication. This hypothesis is strengthened by the observation that TRH is rapidly inactivated by a heterogeneously distributed ecto-enzyme which exhibits a high degree of substrate specificity. Moreover, in the rat, the activity of the membrane-bound TRH-degrading enzyme of the anterior pituitary is found to be stringently controlled by thyroid hormones and estrogens. In contrast, the activity of the TRH-degrading brain enzyme is neither influenced by thyroid hormones nor estrogens. These data indicate that the TRH-degrading brain enzyme serves specialized functions for the transmission of TRH signals and apparently represents the peptidergic equivalent to acetylcholine esterase, whereas the membrane-bound adenohypophyseal TRH-degrading enzyme itself fulfills a biologically important control function within feedback-regulatory mechanisms.     

Actions of releasing factors on isolated secretory granules mediated by calcium.

Synthetic TRH, crude hypothalamic extract and partially purified prolactin releasing factor stimulated prolactin and growth hormone release from isolated secretory granules. Somatostatin and partially purified prolactin release-inhibiting factor inhibited release of both hormones. Calcium promoted hormone release from granules; its releasing action was potentiated by TRH and ionophore A23187 but reduced by somatostatin.     

Sperm motility inhibiting activity of a phytosterol from Alstonia macrophylla Wall ex A. DC. leaf extract: a tribal medicine

The role of methanolic extract and n-butanol fraction of A. macrophylla leaves was investigated on the forward motility of goat spermatozoa. The methanol extract (600 micro/g/ml) and one n-butanol fraction (Fraction A; 100 microg/ml) showed marked inhibition of sperm forward motility, tested by microscopic and spectrophotometric methods. Approximately, 50-60% of the spermatozoa lost their motility when treated with 600 microg/ml of methanol extract or 100 microg/ml of Fraction A. The Fraction A at 400 microg/ml concentration showed complete inhibition of sperm forward motility at 0 min. The inhibitory activity increased with the increasing concentrations of the fraction. The motility inhibitory activity of the Fraction A was stable to heat treatment at 100 degrees C for 2 min. The compound showed high inhibitory effect in the pH range 6.7-7.6. Fraction A also showed high efficacy for inhibiting human sperm motility, assessed by the microscopic method. The phytochemical analysis of methanolic extract of A. macrophylla leaves revealed the presence of sterols, triterpene, flavonoid, alkaloid, tannin and reducing sugar, while the Fraction A contains beta-sitosterol, a common phytosterol. The results demonstrate that Fraction A (beta-sitosterol) is a potent inhibitor of sperm motility and thus it has the potential to serve as a vaginal contraceptive.     

Neuropeptide-specific peptidases: does brain contain a specific TRH-degrading enzyme?

The particulate fraction of brain homogenates contains an enzyme that cleaves the pyroglutamyl-histidyl bond of thyrotropin-releasing hormone (TRH) but is clearly distinct from the more widely distributed pyroglutamyl peptidase (EC 3.4.19.3). This particulate enzyme is highly localized to brain where it is found on synaptosomal membranes. It exhibits an unusual degree of substrate specificity. For example, it does not cleave the pyroglutamyl-histidyl bond of luteinizing hormone-releasing hormone (LHRH) or the pyroglutamyl histidyl bond of the chromogenic substrate pyroglutamyl-histidyl-2-naphthylamide. Evidence is reviewed supporting the possibility that this enzyme, first detected in serum and originally referred to as "thyroliberinase", may be the first neuropeptide-specific peptidase to be characterized.     

Isolation and Identification of Nigragillin as a Insecticidal Metabolite Produced by a Aspergillus niger

Arginine decarboxylase (E.C. 4.1.1) after purification from rice seedlings was separated into fractions A (MW 88000) and B (MW 174000) by gel chromatography. Fraction B was much more active than A. After DEAE cellulose chromatography, the active fraction of the enzyme (B) was purified to homogeneity, which appeared as a single band in gel electrophoresis. The optimal pH and temperature for the enzyme were 8.0 and 45°C, respectively. The enzyme followed typical Michaelis–Menten kinetics with a Km value of 0.28 mm. It had no dependence on a metal, and consisted of 16 amino acids of which proline was prominent. Pyridoxal-5-phosphate acted as a co-factor of the enzyme. The enzyme activity was inhibited by various amines and inhibitors, of which the highest inhibition was obtained with spermine and hydroxylamine. The plant hormones played a vital role in regulating the activity of the enzyme which was promoted by kinetin and inhibited by abscisic acid.     

Purification and characterization of a thyrotropin-releasing hormone deamidase from rat brain.

This report describes the purification of a rat brain thyrotropin-releasing hormone (TRH) deamidating enzyme to apparent homogeneity. Criteria for purity include sodium dodecyl sulfate and disc gel electrophoresis, as well as isoelectric focusing (pI = 4.5). Enzyme purification was facilitated by development of a rapid and sensitive continuous assay using the substrate L-pyroglutamyl-Nim-benzylhistidyl-L-prolyl-beta-naphthylamide, which, upon hydrolysis of the naphthylamide, results in the appearance of the fluorescent product, beta-naphthylamine (beta NA). With this substrate the homogeneous enzyme had a specific activity of 14.5 mumol of beta NA min-1 mg-1. The only peptide product formed was shown to be L-pyroglutamyl-Nim-benzylhistidyl-L-proline. Hydrolysis of [L-prolyl-2,3-3H]TRH was shown to yield L-pyro-glutamyl-L-histidyl-L-proline as the only radiolabeled product. Characterization of the brain deamidase by gel filtration chromatography and sodium dodecyl sulfate gel electrophoresis indicated that the enzyme consists of a single polypeptide chain having molecular weights of 70,000 and 73,500, respectively. Rat brain TRH deamidase has an apparent Km of 34 micron, and a pH optimum between 7 and 8 using L-pyroglutamyl-Nim-benzylhistidyl-L-prolyl-beta-naphthylamide as a substrate. With this substrate, TRH was shown to be a competitive inhibitor with an apparent Ki of 120 +/- 20 micron.     

Peptidase inactivation of hypothalamic releasing hormones.

With the structural characterization of the hypothalamic hormones, luteinizing hormone-releasing hormone (LH-RH), thyrotrophin-releasing (TRH), melanocyte-stimulating hormone release-inhibiting hormine (MIH), and growth hormone release-inhibiting hormone, (GH-RIH or somatostatin), it has been possible to investigate their enzymic inactivation by peptidases which are present at various sites in the body. Enzymes may play an important part in the control of polypeptide hormone levels and the peptidases acting on these four hypothalamic hormones may regulate the amount of TRH, LH-RH, MIH and somatostatin released from the hypothalamus, or their action at the level of the pituitary and their removal from the circulation. By studying the peptidase enzymes, further information may be obtained on the physiological mechanisms controlling the secretion and actions of hypothalamic hormones, as well as on the design of analogues with increased or competitive activity.     

Novel enzyme immunoassay for thyrotropin-releasing hormone using N-(4-diazophenyl)maleimide as a coupling agent

A novel enzyme immunoassay (EIA) for thyrotropin-releasing hormone (TRH) was developed which used N-(4-diazophenyl)maleimide (DPM) as a new heterobifunctional agent capable of cross-linking TRH to mercaptosuccinyl bovine serum albumin and to beta-D-galactosidase. The resulting conjugates act as the immunogen producing anti-TRH serum in rabbits and the enzyme marker of TRH in the EIA, respectively. This EIA with a double-antibody technique was sensitive and reproducible in measuring TRH at concentrations as low as 50 pg per tube, and monospecific to the hormone showing no cross-reactivity with the hormone analogue L-pGlu-L-His-L-Pro and TRH constituents. Using this assay, the distribution of immunoreactive TRH in the brain was determined easily in rats. The use of DPM should provide a valuable new method for developing EIA hitherto possible for other peptide hormones containing neither a free carboxy nor a free amino group, using imidazole, phenolic, and indole group(s) of the amino acid as a reaction site.     

Biological activities of thionated thyrotropin-releasing hormone analogs.

Analogs of thyrotropin-releasing hormone (Glp-His-Pro-NH2, TRH) have been prepared which contain thioamide moieties in the pyroglutamic acid ring, the carboxyamide proline terminus, and in both positions (dithio). These compounds have been tested for TSH-releasing activities (in vitro and in vivo), and for binding to TRH receptors in rat pituitary and cortex. The monothionated analogs showed no significant differences in TSH-releasing potency from TRH either in vitro or in vivo. However, with two thioamide replacements the potency decreases about 50%. Significantly, in terms of receptor selectivity, thionation has resulted in differentiation between brain receptors (pituitary and cortex). The Pro psi[CSNH2] and dithio analogs were more selective (higher affinity to pituitary receptors) than the parent hormone, while the analog containing a thioamide replacement in the pyroglutamyl ring had lower affinity and was not selective. These results suggest that the subtle exchange of sulphur for oxygen can have an important impact on both receptor selectivity and affinity within a biologically active peptide.     

Neuropeptide inhibition of voltage-gated calcium channels mediated by mobilization of intracellular calcium.

Many neurotransmitters and hormones regulate secretion from endocrine cells and neurons by modulating voltage-gated Ca2+ channels. One proposed mechanism of neurotransmitter inhibition involves protein kinase C, activated by diacylglycerol, a product of phosphatidyl-inositol inositol hydrolysis. Here we show that thyrotropin-releasing hormone (TRH), a neuropeptide that modulates hormone secretion from pituitary tumor cells, inhibits Ca2+ channels via the other limb of the phosphatidylinositol signaling system: TRH causes inositol trisphosphate-triggered Ca2+ release from intracellular organelles, thus causing Ca2(+)-dependent inactivation of Ca2+ channels. Elevation of intracellular Ca2+ concentration is coincident with the onset of TRH-induced inhibition and is necessary and sufficient for its occurrence. The inhibition is blocked by introducing Ca2+ buffers into cells and mimicked by a variety of agents that mobilize Ca2+. Treatments that suppress protein kinase C have no effect on the inhibition. Hence inactivation of Ca2+ channels occurs not only as a result of Ca2+ influx through plasma membrane channels, but also via neurotransmitter-induced Ca2+ mobilization. This phenomenon may be common but overlooked because of the routine use of Ca2+ buffers in patch-clamp electrodes.     

The histidine-rich glycoprotein of serum has a domain rich in histidine, proline, and glycine that binds heme and metals

Histidine-rich glycoprotein (HRG) from rabbit serum was digested with plasmin, reduced, and carboxymethylated, and the fragments produced were resolved by reverse-phase high-performance liquid chromatography. Several peptide fractions were obtained that contain unusually high contents of histidine, proline, and glycine. One His-Pro-Gly-rich peptide (apparent Mr 30 000) was obtained in sufficient yield and purity for further study. This peptide is 29 mol % histidine, 37% proline, and 16% glycine, indicating that most of these three amino acids are located in one region of HRG. The peptide contains 9% by weight carbohydrate and is devoid of tyrosine or tryptophan. The far-ultraviolet circular dichroism spectrum of the peptide has a minimum at 203 nm, indicating that the peptide contains polyproline II helical sections. The peptide represents a binding domain of HRG since it retains much of the ability of intact HRG to bind heme and metals including Zn2+, Ni2+, and Cu2+. As with the parent HRG molecule, interaction of the peptide with heme and metals is dependent on pH and intact histidine residues.     

Demonstration of bovine brain calmodulin-dependent cyclic nucleotide phosphodiesterase isozymes by monoclonal antibodies

Calmodulin-dependent cyclic nucleotide phosphodiesterase from bovine brain is found to be composed of two distinct subunits, 60,000- and 63,000-dalton polypeptides. Peptide mapping of the subunits by partial proteolysis demonstrated that the 60-kDa polypeptide is not derived from the 63-kDa species. The interaction of the enzyme with three monoclonal antibodies, A6, C1, and A2, and the analysis of immunocomplexes by sucrose density gradient centrifugation revealed that calmodulin-dependent cyclic nucleotide phosphodiesterase exists in three different forms, i.e. (a) homodiamer of 60-kDa, (b) heterodimer of 60- and 63-kDa, and (c) homodimer of 63-kDa. A6 antibody reacts with both 60- and 63-kDa polypeptides indicating that they are immunologically related. C1 and A2 antibodies react with only 60-kDa polypeptide species. By using C1 Sepharose 4B affinity column chromatography, the 63-kDa homodimer which did not bind to the column (Fraction I) was separated from the 60-kDa polypeptide containing isozymes (the heterodimer and the 60-kDa homodimer) which were retained on the column and later eluted as a mixture (Fraction II). Fraction I, the 63-kDa homodimer enzyme, has higher Vmax toward cGMP as substrate than cAMP whereas the opposite was found with Fraction II. The specific activity of Fraction II enzyme toward cAMP was approximately 500 mumol/min/mg, the highest value ever reported for brain calmodulin-dependent cyclic nucleotide phosphodiesterase preparations.     

Induction of lesions in deoxyribonucleic acid by low molecular weight substances from normal and colicin E2-treated cells of Escherichia coli.

A fraction containing a variety of low molecular weight substances was extracted into 80% aqueous acetone from both a colicin E2-treated cell culture of Escherichia coli and an untreated one. The extract was divided into five fractions by Sephadex G15 chromatography. One of them, Fraction B, was separated into three subfractions by Sephadex G10 chromatography. Two subfractions, Fraction BI and Fraction BII, were further fractionated by several chromatographic systems. DNA was incubated with an aliquot from each of these fractions and was then analyzed by sedimentation in an alkaline sucrose density gradient. The activity which caused a decrease in the sedimentation coefficient of the DNA was found in some of these fractions. The activity from colicin E2-treated cells was compared with that from untreated ones. It was revealed that colicin E2 induces some increases in the activity toward DNA in one of the subfractions, Fraction BI, and also causes the appearance of a new species in another fraction, Fraction BII, which potentiates the activity in Fraction BI. These colicin E2-induced changes appeared at 5 min after the addition of colicin E2. The possible significance of such reactions for the action of colicin E2 are discussed.     

A multicomponent protein of a fission yeast that promotes joint molecule formation from homologous DNAs.

We developed a quantitative assay ("homologous pairing gel assay") adequate for the purification of the activity promoting the formation of joint molecules, an intermediate of homologous recombination ("homologous pairing"). With this assay, one can measure the extent of homologous pairing between a single-stranded DNA and a strand of 3H-labeled double-stranded DNA by crude enzyme preparations. Since the total activity did not significantly change during the sporulation process, we tried to purify the activity from a whole cell extract of mitotic cells of a fission yeast, (Schizosaccharomyces pombe). Through quantitative assaying of a single fraction or of mixed fractions, we obtained three fractions, all of which were required for the maximum level of the ATP-independent homologous pairing: Fractions 65, 100, and 30. In Fractions 100 and 30, polypeptides of approximately 100 and approximately 30 kDa (the 100- and 30-kDa polypeptides), respectively, were the sole detectable components. Fraction 65 contained a polypeptide of approximately 65 kDa (the 65-kDa polypeptide) as the major component and also small amounts of the 30- and 100-kDa polypeptides. Fraction 65 by itself promoted homologous pairing, but the reaction was saturated at a level of approximately 20% of the maximum level achieved with the recA protein. Even when added in excess, Fraction 30 or 100 alone did not promote detectable homologous pairing. A mixture of Fractions 65 and 100 at a rather strict optimum ratio only promoted homologous pairing, the level being 50-70% that with the recA protein, suggesting a stoichiometric complex of these polypeptides as the active form. Fraction 30 alone did not enhance the reaction with Fraction 65, but stimulated homologous pairing promoted by the optimum mixture of Fractions 65 and 100 to the maximum level achieved with the recA protein. Therefore, the homologous pairing-promoting protein from the fission yeast is likely to be a multicomponent protein.