Post-translational modifications of pro-opiomelanocrtin related hormones in medaka pituitary based on mass spectrometric analyses

Direct tissue matrix-assisted laser desorption ionization with time-of-flight mass spectrometry analysis provides a selective detection of mass profile for the peptides contained into cell secretory granules. By this mass spectrometry with slice of pituitary, two novel molecular forms of pro-opimelanocrtin related hormone were found in the orange-red strain medaka (Oryzias latipes var.). The structures of [N,O-diacetyl Serine¹, O-acetyl Serine³]-α-melanocyte-stimulating hormone (MSH) and [hydroxyproline¹⁵]-β-MSH, together with [phosphoserine¹⁵]-corticotropin-like intermediate lobe peptide, were determined for the first time using a collision-induced dissociation with electrospray ionization mass spectrometry. A combination of mass spectrometry analyses is thus a powerful tool to lead to the elucidation of the post-translational processing from the pre-prohormone.     

Combined expression of different hormone genes in single cells of normal rat and mouse pituitary

Cells displaying combined expression of different pituitary hormone genes (further referred to as 'multi-hormone mRNA cells') were identified in normal rat and mouse pituitary by single cell RT-PCR. These cells do not seem to produce or store all the respective hormones the mRNAs encode for. The cells are already developed at day 16 of embryonic life (E16) in the mouse. Different peptides, such as gamma3-melanocyte-stimulating hormone (gamma3-MSH) and gonadotropin-releasing hormone (GnRH), affect different subsets of these cells. In culture, estrogen and GnRH increase the number of 'multi-hormone mRNA cells' that contain prolactin (PRL) mRNA or mRNA of the alpha-subunit of the glycoprotein hormones (alpha-GSU) but not the number of 'multi-hormone mRNA cells' not containing PRL or alpha-GSU mRNA. 'Multi-hormone mRNA cells' may function as 'reserve cells' in which a particular hormone mRNA may be translated under a particular physiological condition demanding a rapid increase of that hormone.     

Studies on the metabolic interactions between 4-methylpyrazole and methanol using the monkey as an animal model

Bovine adrenal chromaffin granules have been shown to contain [Met]enkephalin and [Leu]enkephalin and at least seven other small peptides that exhibit specific binding to opiate receptors. Six of these peptides have been characterized and their structures established as (O)-[Met]enkephalin, [Met]enkephalin-Arg⁶-Arg⁷, [Met]enkephalin-Lys⁶, [Met]enkephalin-Arg⁶, [Leu]enkephalin-Arg⁶, and [Met]enkephalin-Arg⁶-Phe⁷. Many of these hexa- and heptapeptides are also present in bovine and human brain. It is suggested that the presence of these peptides in a tissue is evidence of a common biosynthetic pathway of the enkephalins from a large precursor protein.     

Translation of mengovirus RNA in Ehrlich ascites cell extracts

Mengovirus RNA was translated in Ehrlich ascites cell extracts using as radioactive precursors f[³⁵S]Met tRNA_F^Met and [³⁵S]Met tRNA_M^Met to label the products in N-terminal and internal positions, respectively. Tryptic peptides were compared with those derived from purified [³⁵S]Met-labeled mengovirus. The results indicate that the sequences corresponding to the viral coat polypeptides are preceded by a short “lead-in” peptide which is probably removed by a cleavage process in infected cells.     

Amino-acid Sequence of Human Placental Lactogen

AN earlier report from this laboratory¹ described striking homologies in the amino-acid composition of the peptides obtained from trypsin cleavage of human placental lactogen (HPL) and human growth hormone (HGH) and established a firm biochemical basis for the similarities in biological and immunological activity of the two hormones. The intrachain disulphide bonds were placed in similar locations and the carboxyl-terminal amino-acid sequences were shown to be identical except for one out of fourteen residues². In other studies^3,4, the amino-terminal sequence was determined and noted to be homologous for eleven of the first seventeen residues. We now report the complete amino-acid sequence of HPL and a comparison with the revised structure for HGH recently reported⁴. These two protein hormones are strikingly similar in structure, being identical in 85% of the corresponding positions. Of twenty-eight amino-acid substitutions, twenty-five are favoured changes and all but two of the twenty-five can be explained in terms of a single base change in the triplet codon⁵.     

Characterization of the rabbit renal receptor for native parathyroid hormone employing a radioligand purified by reversed-phase liquid chromatography

Iodinated native bovine parathyroid hormone (bPTH(1-84)) was separated from uniodinated hormone by reversed-phase liquid chromatography techniques after lactoperoxidase labeling. Analysis of iodinated residues after enzymatic digestion indicated that the major labeled product was largely monoiodinated on the sole tyrosine residue. This material retained full bioactivity in an in vitro renal adenylate cyclase assay. Binding of 125I-bPTH(1-84) to rabbit renal membranes at 4 degrees C was proportional to membrane protein concentration and was saturable and dissociable. Radioligand binding was inhibited by concentrations of unlabeled bPTH(1-84) required to stimulate adenylate cyclase in the same membrane preparation but was not inhibited by non-PTH peptides other than adrenocorticotropin at high concentrations (greater than 10 microM). Synthetic NH2-terminal analogues of bPTH(1-84) all elicited approximately equivalent inhibition of radioligand binding which was, however, less potent than unlabeled bPTH(1-84), suggesting a role for the carboxyl region of the molecule in the interaction of bPTH(1-84) with its receptor. Activity of the NH2-terminal agonists was similar to bPTH(1-84) in stimulating adenylate cyclase. Although substitution in sequence position one, of serine in human PTH(1-34) for alanine in bPTH(1-34), reduced activity in the adenylate cyclase assay, inhibition of 125I-bPTH(1-84) binding by both peptides and by an analogue of bPTH(3-34) was equivalent, consistent with a minimal contribution of the first 2 residues for receptor binding of the NH2-terminal region of PTH. The results illustrate the utility of the radiolabeled preparation of native bPTH we have developed and emphasize the importance of probing the PTH receptor with an intact hormone to maximize information concerning the mechanism of PTH action.     

Quantitative aspects of hormone-receptor interactions of high affinity: Effect of receptor concentration and measurement of dissociation constants of labeled and unlabeled hormones

It is demonstrated that because of limitations in the magnitude of the specific activity of radiolabeled hormone derivatives, direct binding studies of hormone-receptor interactions of high affinity (10^?9–10^?11 M, depending on whether ³H- or ¹²³I-labeled hormones are used) will be subject to artifactual distortions due to the need to utilize high concentrations of the receptor. If the concentration of the receptor is not ten times lower than the true affinity constant, the apparent dissociation constant obtained from direct concentration binding curves will vary as a linear function of the receptor concentration. In addition, at high receptor concentrations saturability becomes difficult to demonstrate experimentally and the binding data yield apparently non-hyperbolic, sigmoidal curves which can be mistakenly interpreted to depict cooperative interactions. Similar artifacts related to receptor concentration are predicted for measurements of the hormone concentration dependence of biological processes (e.g. activation of adenylate cyclase, transport processes, etc.). Methods for detecting these effects, and correctly measuring affinities for labeled and unlabeled hormones under these conditions, are described. The implications for measuring the binding properties of hormone-receptor interactions are discussed, especially in reference to studies of the comparative analysis of receptor function in altered metabolic states and to studies relating the biological and binding properties of hormones.     

Synthesis of tritiated rat insulin with high specific activity: A method for radiolabeling the active site of insulin

Insulin was synthesized by rat islets from tritiated amino acids under conditions designed to achieve high specific activity. Islets were isolated by the collagenase method. Stores of unlabeled insulin were depleted by culturing them for 40 hours in the presence of 3-isobutyl-1-methylxanthine. The islets were then incubated for 22 hours in the presence of [³H]Isoleucine or [³H]Phenylalanine. These amino acids were chosen because they are specific markers from the A and B chains of insulin respectively. Labeled insulin was extracted from the islets and purified by gel filtration. Its biological activity was indistinguishable from monoiodinated insulin as assessed by binding to receptors on cultured human lymphocytes and by precipitation by anti-insulin antibodies. The specific activity was (18 Ci/mmole) and (37 Ci/mmole) for [³H]Ile and [³H]Phe insulin respectively.     

Isolation of Neurohypophysial Hormones of <Emphasis Type="Italic">Rana temporaria</Emphasis>

THE neurohypophysial hormones of several vertebrates have been identified^1–3, but the nature of the neurohypophysial principles of amphibians is in doubt. Vasotocin (8 Arg-oxytocin) is known to be present, but the oxytocic component could be either oxytocin, mesotocin (8 Ileu-oxytocin) or both^1–7. Since the neutral peptides are difficult to distinguish by chromatographic and pharmacological methods, we chemically analysed the isolated peptides which is necessary to identify the hormones. To prevent any possible contamination with foreign neurohypophysial principles, it is essential to isolate the active components without the use of exogene hormone binding proteins (neurophysins)⁸.     

Studies on the iodination of LH-RH and the biological and immunological activities of the products

Synthetic LH-RH was iodinated by the modified chloramine-T or lactoperoxidase method, using ¹²⁷INa or ¹²⁵INa. The yields of the products, the LH releasing activities of the monoiodinated peptides as well as binding to pituitary membrane fractions were measured. The variation in yield in the four procedures used for iodination was a function of the amount of oxidizing agent. Monoiodinated products obtained by the different procedures possessed comparable LH-releasing activities as well as binding affinity to pituitary plasma membranes.     

The influence of neuropeptides on amine synthesis: Cholecystokinin-8 and neurotensin reduce striatal tyrosine hydroxylase activity

Cholecystokinin-8 and neurotensin, neuropeptides found in relatively high concentrations in some catcholaminergic neurons and/or terminal or cell body areas, reduced native and polyanion-activated rat striatal tyrosine hydroxylase activity, but not that of the enzyme activated by phosphorylating conditions. Substance P, γ₃-melanocyte-stimulating hormone, [d-ala², d-leu⁵]-enkephalin, or thyrotropin-releasing hormone had no effect on the native enzyme or on either activated form. Cholecystokinin-8 and neurotensin may interact with polyanion-activated or membrane bound-activated tyrosine hydroxylase to modulate its activity.     

Purification of [I]-vasoactive intestinal peptide by reverse-phase HPLC

VIP was labeled with sodium ¹²⁵iodide, and ¹²⁵I-VIP was purified by reverse-phase high performance liquid chromatography. Optimal separations of ¹²⁵I-VIP and unlabeled VIP were obtained using two C₁₈-Novapak columns in series and a gradient of acetonitrile in triethylamine phosphate for elution. The specific activity of the ¹²⁵I-VIP was 1.99±0.21 Ci/μmole, approaching the maximum specific activity of monoiodinated VIP (2.26 Ci/μmole). Radioimmunoassay and radioreceptorassay for VIP were more sensitive (2.6-fold, and 2.5-fold, respectively) using ¹²⁵I-VIP purified by HPLC compared to ¹²⁵I-VIP obtained from an open-end cellulose column. These results demonstrate the advantage of preparing purified ¹²⁵I-VIP by HPLC for the accurate assay of VIP and VIP-receptors in tissues and biological fluids.     

Binding and internalization of gold-conjugated somatostatin and growth hormone-releasing hormone in cultured rat somatotropes

Summary The synthetic peptides somatostatin (SRIF) and growth hormone-releasing hormone (GRH) were coupled directly to colloidal gold of different particle sizes. Both conjugates were biologically active in displacing the corresponding radiolabeled hormones from high affinity binding sites in pituitary membranes. Release of growth hormone (GH) from cultured anterior pituitary cells was modulated by both conjugates alone or in combination. Ultrastructural studies were performed with cells incubated at 4° C (2 h) and 37° C (2 min-2 h) with one of the labeled peptides or their combination. Somatotropes were identified by immunostaining with anti-rGH followed by protein A-ferritin, thus obtaining a triple labeling. Both hormone conjugates were internalized in different vesicles in the beginning but accumulated during longer incubation times in the same compartment. The secretory vesicles and the nucleus were not labeled by any hormone conjugate. In contrast to SRIF-gold, the uptake of GRH-gold conjugate decreased with longer incubation times. This effect could be neutralized by simulatenous incubation of the somatotropes with both regulating hormones. Hence, whereas the binding and internalization of SRIF by somatotropes do not seem to be influenced by GRH, the corresponding processes for GRH are stimulated by the presence of SRIF.     

Biodegradable carriers for the sustained release of polypeptides

It is one thing to produce pharmacologically active peptides: it is quite another to formulate them as practical and effective drugs. For many polypeptides, particularly hormones, it is desirable to release the drug continuously at a controlled rate over a period of weeks or even months. This paper describes the development of injectable, biodegradable depot formulations of the highly potent, synthetic analogue of luteinizing hormone releasing hormone, d-Ser (Bu^t)⁶, Azygly¹⁰LHRH, (‘Zoladex’1, ICI 118630) which causes a selective castration-like effect in animals and man which leads to regression of hormone responsive tumours. This technology has been applied successfully to give controlled release formulations of polypeptides having a range of molecular weights.     

Genomics, transcriptomics, and peptidomics of Daphnia pulex neuropeptides and protein hormones

We report 43 novel genes in the water flea Daphnia pulex encoding 73 predicted neuropeptide and protein hormones as partly confirmed by RT-PCR. MALDI-TOF mass spectrometry identified 40 neuropeptides by mass matches and 30 neuropeptides by fragmentation sequencing. Single genes encode adipokinetic hormone, allatostatin-A, allatostatin-B, allatotropin, Ala(7)-CCAP, CCHamide, Arg(7)-corazonin, DENamides, CRF-like (DH52) and calcitonin-like (DH31) diuretic hormones, two ecdysis-triggering hormones, two FIRFamides, one insulin, two alternative splice forms of ion transport peptide (ITP), myosuppressin, neuroparsin, two neuropeptide-F splice forms, three periviscerokinins (but no pyrokinins), pigment dispersing hormone, proctolin, Met(4)-proctolin, short neuropeptide-F, three RYamides, SIFamide, two sulfakinins, and three tachykinins. There are two genes for a preprohormone containing orcomyotropin-like peptides and orcokinins, two genes for N-terminally elongated ITPs, two genes (clustered) for eclosion hormones, two genes (clustered) for bursicons alpha, beta, and two genes (clustered) for glycoproteins GPA2, GPB5, three genes for different allatostatins-C (two of them clustered) and three genes for IGF-related peptides. Detailed comparisons of genes or their products with those from insects and decapod crustaceans revealed that the D. pulex peptides are often closer related to their insect than to their decapod crustacean homologues, confirming that branchiopods, to which Daphnia belongs, are the ancestor group of insects.     

Radiation chemical studies of Gly-Met-Gly in aqueous solution

Abstract

Important biological consequences are related to the reaction of HO^? radicals with methionine (Met). Several fundamental aspects remain to be defined when Met is an amino acid residue incorporated in the interior of peptides and proteins. The present study focuses on Gly-Met-Gly, the simplest peptide where Met is not a terminal residue. The reactions of HO^? with Gly-Met-Gly and its N-acetyl derivative were studied by pulse radiolysis technique. The transient absorption spectra were resolved into contributions from specific components of radical intermediates. Moreover, a detailed product analysis is provided for the first time for Met-containing peptides in radiolytic studies to support the mechanistic proposal. By parallel radiolytical and electrochemical reactions and consequent product identification, the formation of sulfoxide attributed to the direct HO^? radical attack on the sulfide functionality of the Met residue could be excluded, with the in situ generated hydrogen peroxide responsible for this oxidation. LC–MS and high resolution MS/MS were powerful analytical tools to envisage the structures of five products, thus allowing to complete the mechanistic picture of the overall Met-containing peptide reactivity.     

Methoxinine – an alternative stable amino acid substitute for oxidation‐sensitive methionine in radiolabelled peptide conjugates

Radiolabelled peptides with high specificity and affinity towards receptors that are overexpressed by tumour cells are used in nuclear medicine for the diagnosis (imaging) and therapy of cancer. In some cases, the sequences of peptides under investigations contain methionine (Met), an amino acid prone to oxidation during radiolabelling procedures. The formation of oxidative side products can affect the purity of the final radiopharmaceutical product and/or impair its specificity and affinity towards the corresponding receptor. The replacement of Met with oxidation resistant amino acid analogues, for example, norleucine (Nle), can provide a solution. While this approach has been applied successfully to different radiolabelled peptides, a Met → Nle switch only preserves the length of the amino acid side chain important for hydrophobic interactions but not its hydrogen‐bonding properties. We report here the use of methoxinine (Mox), a non‐canonical amino acid that resembles more closely the electronic properties of Met in comparison to Nle. Specifically, we replaced Met¹⁵ by Mox¹⁵ and Nle¹⁵ in the binding sequence of a radiometal‐labelled human gastrin derivative [d ‐Glu¹⁰]HG(10‐17), named MG11 (d ‐Glu‐Ala‐Tyr‐Gly‐Trp‐Met‐Asp‐Phe‐NH₂). A comparison of the physicochemical properties of ¹⁷⁷Lu‐DOTA[ X ¹⁵]MG11 ( X = Met, Nle, Mox) in vitro (cell internalization/externalization properties, receptor affinity (IC₅₀), blood plasma stability and logD) showed that Mox indeed represents a suitable, oxidation‐stable amino acid substitute of Met in radiolabelled peptide conjugates. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

Preparation and characterization of radioactive monoiodotyrosine and diiodotyrosine derivatives of parathyroid hormone

Highly purified native parathyroid hormone was iodinated by the enzymatic method and separated from unlabeled hormone by isocratic HPLC. The separation system used also resolved iodohistidine, monoiodotyrosine, and diiodotyrosine forms of the hormone from one another. A simplified procedure for direct bioassay of the carrier-free, high specific activity, mono- and diiodinated parathyroid hormone (PTH) by the renal membrane adenylyl cyclase method was also developed. Both labeled forms of the hormone are very potent in this assay, but the iodinated forms appeared to give a lower V_max than the native hormone. The methods for iodination, separation and biological characterization of this PTH tracer are exceptionally facile, inexpensive, and convenient.     

Changes in pituitary hormone levels induced by met-enkephalin in man—The role of dopamine

The interaction of dopamine with the effects of the opiate agonist peptide D-Ala²-MePhe⁴-met-enkephalin-O-o1 (DAMME) on anterior pituitary hormone secretion was investigated in normal male subjects. DAMME produced clear elevations in prolactin, growth hormone and thyroid-stimulating hormone, while inhibiting the release of luteinising hormone and cortisol. There was no change in follicle stimulating hormone. The elevations in prolactin and TSH were enhanced by the dopamine antagonist, domperidone, and blocked by an infusion of dopamine. Neither dopamine nor domperidone modulated the changes in growth hormone, luteinising hormone or cortisol. The data are comptible with the association of the release of prolactin and TSH by opiate peptides with decreased hypothalamic dopaminergic activity; changes in the other anterior pituitary hormones seem to involve different mechanisms.