The Posttranslational Processing of β-Endorphin in Human Hypothalamus

beta-Endorphin is posttranslationally processed to six derivatives, which, although structurally similar, produce distinctly different biological effects. beta-Endorphin 1-31 is a potent opioid receptor agonist, but beta-endorphin 1-27 exhibits antagonist properties, and beta-endorphin 1-26 and the alpha-N-acetyl derivatives of all three peptides lack opioid receptor activity. In the present study, we identified the beta-endorphin peptides synthesized in human hypothalamus using cation exchange HPLC. First, we tested whether postmortem changes occur by storing rat hypothalami at 4 degrees C. This demonstrated that relative amounts of the six beta-endorphin forms did not change for up to 24 h, although total beta-endorphin immunoreactivity significantly declined after 6 h. HPLC analysis of human hypothalami revealed that beta-endorphin 1-31 was the principal form, constituting 58.4 +/- 5.4% of total immunoreactivity. Substantial amounts of beta-endorphin 1-27 (13.4 +/- 1.2%) and beta-endorphin 1-26 (13.1 +/- 1.6%) were also present, but alpha-N-acetylated forms were quantitatively minor, each comprising approximately 5% of total beta-endorphin. A similar processing pattern occurred in preoptic and suprachiasmatic areas of the hypothalamus. These results show that, despite differences in primary sequence, beta-endorphin is processed similarly in both rat and human hypothalamus. Opiate-active beta-endorphin 1-31 is the principal form in both species.     

Effect of the A118G polymorphism on binding affinity, potency and agonist-mediated endocytosis, desensitization, and resensitization of the human mu-opioid receptor

The most prevalent single-nucleotide polymorphism (SNP) A118G in the human mu-opioid receptor gene predicts an amino acid change from an asparagine residue to an aspartatic residue in amino acid position 40. This N40D mutation, which has been implicated in the development of opioid addiction, was previously reported to result in an increased beta-endorphin binding affinity and a decreased potency of morphine-6-glucuronide. Therefore, in the present study we have investigated whether this mutation might affect the binding affinity, potency, and/or the agonist-induced desensitization, internalization and resensitization of the human mu-opioid receptor stably expressed in human embryonic kidney 293 cells. With the exception of a reduced expression level of N40D compared to human mu-opioid receptor (hMOR) in HEK293 cells, our analyses revealed no marked functional differences between N40D and wild-type receptor. Morphine, morphine-6-glucuronide and beta-endorphin revealed similar binding affinities and potencies for both receptors. Both the N40D-variant receptor and hMOR exhibited robust receptor internalization in the presence of the opioid peptide [d-Ala(2),N-MePhe(4),Glyol(5)]enkephalin (DAMGO) and beta-endorphin but not in response to morphine or morphine-6-glucuronide. After prolonged treatment with morphine, morphine-6-glucuronide or beta-endorphin both receptors showed similiar desensitization time courses. In addition, the receptor resensitization rates were nearly identical for both receptor types.     

Origin of bombesin-like peptides in human fetal lung

Four different forms of bombesin-like immunoreactive peaks were detected in extracts of human fetal lung by the use of reversed-phase high performance liquid chromatography (HPLC). Peaks I, II, III and IV, (increasing retention time), were eluted using a 14-38% of acetonitrile gradient containing 0.1% trifluoroacetic acid (TFA). Peak II was the major material found in the extract of human fetal lung obtained at 16-20 weeks gestation. None of the four compounds contained in the eluted peaks had the same retention time as amphibian bombesin or porcine gastrin releasing peptide (GRP). On reversed-phase HPLC using two different solvent systems TFA or heptafluorobutyric acid (HFBA) as a hydrophobic counter ion, and in gel filtration chromatography, the chromatographic behavior of the main peak (peak II) was the same as that of the carboxyl terminal fragments of GRP, GRP18-27 or GRP19-27. This suggested that the peptide(s) in peak II resembled in composition the carboxy terminal 9 or 10 amino acids of porcine GRP. Following tryptic digestion the material in peak IV was converted to the more polar compound present in peak II. Two other peptide peaks were eluted close to peak II and these were presumed to be a modification of this main peak. One of the possible biosynthetic steps in the formation of bombesin-like peptides in human fetal lung could be a tryptic conversion of a less polar peptide to a more polar form (peak IV to II).     

A modified Arg-Asp-Val (RDV) peptide derived during the synthesis of Arg-Glu-Asp-Val (REDV), a tetrapeptide derived from an alternatively spliced site in fibronectin, inhibits the binding of fibrinogen, fibronectin, von Willebrand factor and vitronectin to activated platelets.

Characterization of a side-product obtained during the synthesis of Arg-Glu-Asp-Val (REDV) with inhibitory activity in thrombin-activated platelet aggregation was carried out. The semipreparative column fractionation of REDV peptide was rechromatographed on an analytical HPLC column and revealed two peaks which were re-tested for inhibitory activity. Using amino acid analysis with sequencing and fast atom bombardment mass spectrometry (FABMS), the first peak was determined to be REDV with molecular mass of 517 Da, and the second peak was determined to be a modified RDV with a mass of 608 Da. The modified RDV peptide inhibited thrombin-induced platelet aggregation with an IC50 of 200 microM, and complete inhibition occurred at 600 microM. However, the REDV peptide did not inhibit platelet aggregation up to 1 mM concentration. The modified RDV peptide eluted platelet glycoprotein IIb-IIIa complex that had been bound to GRGDSP-agarose. These studies show that the modified RDV peptide interacts with the platelet glycoprotein IIb-IIIa complex. Based on the collision-induced dissociation (CID) mass spectral data analysis, the modified RDV peptide has been characterized to contain an N-terminus blocking group on the Arg residue. The origin of this blocking group is presumed to have originated from decomposition products of the phenylacetamidomethyl (PAM) resin used in the solid-phase synthesis of the target peptide Arg-Glu-Asp-Val.     

Purification and characterization of the central segment of prothymosin-alpha: methodology for handling highly acidic peptides.

Prothymosin-alpha is a highly acidic protein consisting of 110 amino acids. The central segment of this protein, residues 51-89, is thought to be involved in metal binding which may be necessary for its physiological function. To carry out studies of this peptide, this central segment was synthesized in a linear fashion using Fmoc-based methods on rink amide MBHA resin. However, this peptide could not be purified with the typical straightforward approach of RP HPLC followed by negative mode electrospray ionization mass spectrometry (ESI-MS). This was attributed to the high proportion of acidic residues: 26 out of the 39 residues are aspartic and glutamic acids. The acidity of the peptide prevented retention on the RP HPLC column. Additionally, the ability of the highly negatively charged peptide to retain sodium ions prevented molecular weight determination with ESI-MS. A systematic approach to the purification of this highly acidic peptide was undertaken. Ultimately, strong anion exchange chromatography was used to purify the peptide. Extensive desalting using dialysis was required prior to ESI-MS, and the choice of the buffer proved to be critical. In the end, a purification method was devised that yielded a highly purified peptide and is readily compatible with analysis by ESI-MS.     

The chemical identity of the immunoreactive LHRH-like peptide biosynthesized in the human placenta

Freshly obtained human placental trophoblasts were minced and pulselabeled for 30 min at 37°C with tritiated L-Tyrosine. After homogenisation, the crude extract was centrifuged and deproteinized with 10% TCA. The supernatant was defatted and the peptides concentrated through hydrophobic binding on ODS-silica cartridges. The bound, crude peptide extract was eluted and subjected to gradient, reverse-phase High Performance Liquid Chromatography. The fractions corresponding to the absorption peak of reference, synthetic LHRH were collected and extensively purified to radioactive homogeneity by further multiple HPLC. After digestion with pyroglutamate aminopeptidase, the resulting nonapeptide was manually sequenced by dansyl-Edman degradation. All the incorporated radioactivity was found to reside exclusively in residue number 4 of the nonapeptide; thus establishing for the first time the primary sequence of biosynthetic placental LHRH as: pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH₂, identical to its hypothalamic counterpart.     

虎纹捕鸟蛛毒素-Ⅰ-(HWTX-Ⅰ)28肽类似物的化学合成与活性鉴定

虎纹捕鸟蛛毒素-(huwentoxin-,HWTX-)是从我国虎纹捕鸟蛛毒素中分离纯化得到的一种多肽类神经毒素.该多肽分子由33个氨基酸残基组成,含三对二硫键.其一级结构和三级结构均已测定.弄清该毒素的活性部位,是研究其结构功能关系的基础.用固相Fmoc化学合成的方法,合成了虎纹捕鸟蛛-的28肽类似物.该突变体去掉了天然毒素分子N端的Alal和C端的Lys30-Trp31-Lys32-Leu33共5个残基.用氧化还原型谷胱甘肽法完成二硫键配对,并用HPLC进行纯化,所得突变体与天然HWTX-的紫外光谱相似.质谱鉴定确认合成产物正确,分子量为3124,浓度为1×10-5g/ml的突变体能可逆阻断小白鼠膈神经-膈肌的接头传递,阻断时间为60～70min.与天然毒素比较,活性有所下降.结果说明HWTX-的N端、C端残基对其生物活性有一定影响,但不是位于活性中心的重要残基.由结果推测,虎纹捕鸟蛛毒素-的活性中心位于该分子中连接β-折叠的回环区     

Optimization of immobilized enzyme hydrolysis combined with high-performance liquid chromatography/thermospray mass spectrometry for the determination of neuropeptides

Peptidases, including chymotrypsin, thermolysin, trypsin, V8 protease, and carboxypeptidases A, B, and Y, were immobilized for use in conjunction with HPLC/thermospray MS for the analysis of neuropeptides. The optimal operating conditions for each immobilized enzyme bioreactor were determined. Optimal hydrolysis usually occurred at the highest percentage of aqueous solution in the mobile phase at pH 7-8 and 40-50 degrees C. Often post-HPLC column addition of aqueous solutions before the bioreactor could improve activity and thermospray sensitivity without changing the HPLC separation. Enzymatic hydrolysis requirements were compatible under conditions for HPLC separation and thermospray MS detection of the selected neuropeptides. Synthetic alpha-, beta-, and gamma-endorphins were the primary neuropeptides used to evaluate on-line immobilized enzyme bioreactor/MS. HPLC followed by peptidase hydrolysis produced characteristic hydrolysis products for confirming the peptides' identity using thermospray MS detection. Furthermore, the peptide formed from enzymatic hydrolysis resulted in a MS ion current 10-40 times higher than that of the [M + 2H]2+ ion for unhydrolyzed beta-endorphin. The increased sensitivity achieved for detecting the hydrolysis products permits detection and quantitation of synthetic peptides down to 800 fmol.     

Identification of a novel amino acid, o-bromo-L-phenylalanine, in egg-associated peptides that activate spermatozoa

Eight sperm-activating peptides containing a novel amino acid were isolated from the egg jelly of the sea urchin Tripneustes gratilla. Accurate mass measurement of the peptide in FAB mass spectrometry showed that the mass of the novel amino acid residue was 224.978. On the basis of the isotopic ion distribution and the degree of unsaturation, the mass value indicated that the elemental composition of the amino acid residue was C9H8O1N1Br1, suggesting that the novel amino acid was bromophenylalanine. Proton NMR spectroscopy, amino acid analysis, and RP-HPLC with three synthetic isomers of bromophenylalanine demonstrated that o-bromophenylalanine was the novel amino acid. Derivatization of the amino acid with Marfey's reagent, (1-fluoro-2,4-dinitrophen-5-yl)-L-alanine amide (FDAA), further indicated that the amino acid was the L-isomer. In other sperm-activating peptides isolated from the egg jelly of the sea urchin, both m- and p-bromophenylalanines were discovered. The presence of m-bromophenylalanine has not been previously reported in natural products, while p-bromophenylalanine is found in theonellamide F, an antifungal bicyclic peptide from a marine sponge.     

Synthesis and characterization of a novel reagent containing dansyl group, which specifically alkylates sulfhydryl group: an example of application for protein chemistry

We have synthesized a novel reagent containing dansyl group, iodoacethyl dansylcadaverine (IADC), which specifically alkylates sulfhydryl groups. The carboxyl group of iodoacetic acid was activated with dicyclohexylcarbodiimide and was condensed with amino group of dansylcadaverine. Purity and chemical structure of IADC was confirmed with mass spectrometry (MS) and NMR. IADC alkylated GSH but not GSSG, which was confirmed by MS. The reactivity of IADC with proteins was also investigated with Western blotting using anti-dansyl antibody. IADC reacted only with sulfhydryl-containing proteins. The specificity of the interaction of IADC with sulfhydryl groups in proteins was confirmed by adding excessive amount of a well-known sulfhydryl-specific reagent, 5, 5'-dithiobis(2-nitrobenzoic acid), which led to a complete inhibition. To show the usefulness of IADC, the cysteines in glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from chicken muscle were modified with this reagent, and GAPDH was then digested by lysyl endopeptidase. The peptides generated from digestion of IADC-incorporated GAPDH were applied to an anti-dansyl immunoaffinity column. The peptide fragments bound and eluted from the column were separated by HPLC, and the amino acid sequence of each peptide was analyzed, and peptide was identified as the one containing a Cys residue(s). These data showed that IADC is a useful reagent to specifically identify the positions of a Cys residue(s) in proteins.     

Purification of a hydrophobic surfactant peptide using high-performance liquid chromatography

A 4- to 6-kDa hydrophobic peptide (SP4-6) was purified from human pulmonary surfactant. Sep Pak Florisil cartridges removed most of the lipids and the 18-kDa peptide. Analytical wide-pore reversed-phase HPLC column separated a single peptide that contained no detectable lipids (less than 1 nmol/2.5 micrograms protein). N-terminal analysis indicated that this peptide was pure, but the N-terminal amino acid was blocked. The peptide was capable of restoring the in vitro surface properties of synthetic phospholipids, which is characteristic of native lung surfactant.     

Characterization of endorphins from the pituitary of the spiny dogfish Squalus acanthias

Opioid-like immunoreactive material was extracted from the pituitary and brain of the Spiny Dogfish Shark Squalus acanthias. The immunoreactive material in the pituitary extracts was purified to apparent homogeneity by reverse phase high performance liquid chromatography and subsequently characterized by amino acid analysis, Edman degradation and fast atom bombardment mass spectrometry. The largest opioid-like peptide isolated contained 30 amino acids and showed 80 percent homology with salmon endorphin-II but less than 50 percent homology with human beta-endorphin. Three structural variants of this molecule were also characterized. These variants were shown to be shorter N-terminal fragments, two of which corresponded to cleavage products at the single basic residues arginine and lysine. Cleavage at a single lysine residue has not been reported for posttranslational processing of beta-endorphin in mammals and could represent a modification seen only in lower vertebrates. The remaining fragment corresponded to a loss of 3 residues from the C-terminus of the parent molecule. No alpha-N-acetylated peptides were detected. These results provide the first unequivocal confirmation of beta-endorphin in an elasmobranch and provide evidence of novel N-terminal variants of beta-endorphin.     

Structural comparison of bovine erythrocyte, brain, and liver NADH-cytochrome b5 reductase by HPLC mapping

NADH-cytochrome b5 reductases purified from bovine erythrocytes and from bovine brain and liver microsomes solubilized with lysosomal protease were subjected to structural analysis by using HPLC mapping, amino acid analysis of the resulting peptides, and NH2-terminal sequence analysis of apoproteins. HPLC maps of the tryptic peptides derived from these enzymes were very similar to each other, and amino acid analysis of the HPLC-separated peptides indicated that the structures of these enzymes are identical except for the NH2-terminal region. The NH2-terminal sequence of the brain enzyme determined by automated Edman degradation was as follows: NH2-Phe-Gln-Arg-Ser-Thr-Pro-Ala-Ile-Thr-Leu-Glu-Asn-Pro-Asp- Ile-Lys-Tyr-Pro-Leu-Arg-Leu-Ile-Asp-Lys-Glu-Val-Ile- This sequence is identical to that of liver enzyme except that the liver enzyme started at the 3rd Arg or 4th Ser. The NH2-terminal amino acid residue of the soluble erythrocyte enzyme was not detected by automated Edman degradation. The sequence analysis of a tryptic peptide from the erythrocyte enzyme indicated that Leu is present before the NH2-terminal Phe of the brain enzyme. The recently reported sequence of the apparently identical protein (Ozols et al. (1985) J. Biol. Chem. 260, 11953-11961) differs in two amino acid assignments from our sequence.     

Detection of N-acetylated forms of beta-endorphin and nonacetylated alpha-MSH in the intermediate pituitary of the toad, Bufo marinus

Steady-state analysis of the acid extracts of the intermediate pituitary of the toad, Bufo marinus, revealed the presence of multiple forms of beta-endorphin and alpha-MSH. Approximately 98% of the immunoreactive beta-endorphin was N-acetylated. The major form of N-acetylated beta-endorphin, which represented 81.5% of the total beta-endorphin recovered from this tissue, had an apparent molecular weight of 1.2 kDa and a net charge of +1 at pH 2.75. Approximately 98% of the immunoreactive alpha-MSH present in the Bufo intermediate pituitary had reverse phase HPLC properties similar to the nonacetylated form of alpha-MSH, ACTH(1-13)amide. These observations are in agreement with studies on the intermediate pituitary of the frog, Xenopus laevis, which have shown that the N-acetylation of alpha-MSH in this species is a cosecretory processing event, whereas the N-acetylation of beta-endorphin is a posttranslational processing event (2, 5, 15). These observations indicate that the N-acetylation of beta-endorphin and alpha-MSH occurs at distinct subcellular sites in intermediate pituitary cells of anuran amphibians. The Bufo intermediate pituitary will serve as a good model system for studying these novel N-acetyltransferase reactions.     

Nucleotide sequence of cDNA for the eclosion hormone of the silkworm, Bombyx mori, and the expression in a brain.

The cDNAs encoding eclosion hormone (EH) of the silkworm, Bombyx mori, were isolated and sequenced. The results showed that the pre-EH molecule contains a 26-amino acid signal peptide and a 62-amino acid mature EH. The deduced amino acid sequence agreed with that previously determined by the peptide analysis. The presence of leucine residue at the carboxyl terminal of EH, which had not been detected directly by the peptide analysis, was proved. Primer extension and Northern hybridization analyses revealed that 0.9 kb mRNA is transcribed and it has a 66-nucleotide non-translated sequence at the 5'-end region. In situ hybridization showed that the EH gene is expressed in two pairs of nuerosecretory cells in the brain of 5th instar larva.     

重组虎纹捕鸟蛛毒素—I在巴氏毕赤酵母中的表达及纯化

虎纹捕鸟蛛毒素I是从虎纹捕鸟蛛粗毒中分离纯化,具有镇痛活性的肽类神经毒素。对巴氏毕赤酵母生产的重组HWTX－I进行多步纯化,首先将分泌到培养上清的rHWTX-I进行90％饱和度的（NH4）2SO4沉淀,再用截留分子量3kD的滤膜脱盐,再用CM阳离子交换层析分离,最后用C18反相层析脱盐纯化,真空干燥后得到的rHWTX-I经Tricine SDS-PAGE,质谱鉴定,氨基酸组成分析,N－端序列测定后活性鉴定,证明已获得高纯度的重组HWTX－I,摇瓶表达量约为80mg/L,约占总分泌量的23．6％,并对摇瓶发酵条件进行了优化,为利用基因工程方法生产HWTX－I的规模化生产及临床应用提供了证据。     

An endogenous peptide that stimulates lanthanum-resistant calcium uptake in vascular tissue

A recent report has described the preparation of an extract from hemolyzed erythrocytes that has a stimulatory effect on lanthanum-resistant calcium uptake by vascular tissue in vitro and a hypertensive effect when injected into normotensive rats. The compound having a stimulatory effect on calcium uptake was further fractionated by molecular sieve and ion exchange chromatography, precipitation with CaCl2, high voltage paper electrophoresis, and high performance liquid chromatography (HPLC). HPLC yielded only a single fraction containing biological activity. This fraction was ninhydrin positive and acid labile. The amino acid composition was as follows: Asp/Asn (1.41), Ser (1.02), Glu/Gly (1.00), and Gly (2.00). Based on the assumption that the compound contains a single glutamic acid or glutamine residue, concentration-response data indicated that only nanomolar amounts of material were necessary to achieve significant stimulation. There was a marked increase in stimulatory activity of the resolubilized compound following calcium precipitation. The compound became inactive or showed a reduction in activity after being applied to a cation exchange column to remove calcium. Subsequent reprecipitation with CaCl2 and resolubilization restored the lost activity. Thus, we conclude that the compound is a small, acidic, calcium-dependent peptide that is extremely potent in stimulating lanthanum-resistant calcium uptake in vascular tissue.     

Isolation and structural characterization of three isoforms of recombinant consensus alpha interferon

Recombinant DNA-derived consensus alpha interferon was expressed in Escherichia coli and purified. Isoelectric focusing of this purified protein indicated the presence of three isoelectric subforms of pI 6.1, 6.0, and 5.7. These three subforms were preparatively separated by isoelectric focusing using Immobiline polyacrylamide gel and did not exhibit apparent differences in biological activity and tertiary structure. The pI 5.7 subform could also be separated from the pI 6.1 and 6.0 subforms by reverse-phase HPLC. Automated N-terminal amino acid sequence analysis of the pI 6.1 and 6.0 subforms yielded sequences corresponding to the methionyl and des-methionyl forms of the protein, respectively. Sequence analysis of the pI 5.7 subform indicated that its N terminus is blocked. To further determine the structure of the blocking moiety in the pI 5.7 subform, a blocked N-terminal tryptic peptide was isolated from HPLC peptide mapping of the S-carboxymethylated derivative. Results obtained from mass spectroscopic and amino acid analyses of this peptide suggest that it is blocked with an acetyl group at the N-terminal cysteine residue.