Pro-enkephalin peptides possessing Met-enkephalin-Arg6-Gly7-Leu8-immunoreactivity in rat CSF, striatum and adrenal gland

Cerebrospinal fluid (CSF) taken from rats implanted with chronic cisternal cannulae and extracts prepared from rat adrenal gland and striatum were subjected to Sephadex G-50 chromatography and HPLC. Fractions were monitored using specific radioimmunoassays (RIA) for the pentapeptide methionine enkephalin (Met-Enk) and methionine enkephalin-Arg6-Gly7-Leu8 (Met-EnkRGL). In rat CSF, striatum and adrenal gland, three Met-EnkRGL-immunoreactive (IR) peaks of Mrs 8000, 5000 and 1000 daltons were detected. The same peaks were also found to possess Met-Enk-immunoreactivity after enzyme digestion of Sephadex G-50 fractions with trypsin and carboxypeptidase B (CPB), suggesting their derivation from proenkephalin. HPLC of the 8K and 5K peaks on a column of Ultrapore RPSC showed them to elute discretely with similar retention times, indicative of hydrophobic peptides of large molecular weight. Their similar hydrophobicities yet significant separation during gel filtration would suggest that the 8K and 5K peptides are structurally closely related yet different with respect to their molecular weights. HPLC of the small molecular weight material from rat striatum and adrenal gland revealed the presence of Met-EnkRGL and Met-EnkRGL sulphoxide in both tissues. In rat striatum Met-Enk and its sulphoxide were also detected. The oxidised pentapeptide was found to be present in rat CSF, together with two unidentified small molecular weight Met-Enk-IR peaks detected without prior enzyme digestion of fractions. The small molecular weight Met-EnkRGL-IR material in rat CSF was found to be comprised of two unknown peptides which were less hydrophobic than Met-EnkRGL and its sulphoxide derivative.     

Four day inhibition of prolyl oligopeptidase causes significant changes in the peptidome of rat brain, liver and kidney

Prolyl oligopeptidase (PREP) cleaves short peptides at the C-side of proline. Although several proline containing neuropeptides have been shown to be efficiently cleaved by PREP in vitro, the actual physiological substrates of this peptidase are still a matter of controversy. The aim of this study was to evaluate the changes in the peptidome of rat tissues caused by a repeated 4-day administration of the potent and specific PREP inhibitor KYP-2047, using our recently developed iTRAQ-based technique. We found tissue-dependent changes in the levels of specific subsets of peptides mainly derived from cytosolic proteins. Particularly in the kidney, where the levels of cytochrome c oxidase were found decreased, many of the altered peptides originated from mitochondrial proteins being involved in energy metabolism. However, in the hypothalamus, we found significant changes in peptides derived from hormone precursors. We could not confirm a role of PREP as the metabolising enzyme for β-endorphin, galanin, octadecaneuropeptide, neuropeptide–glutamic acid–isoleucine, substance P, somatostatin, enkephalin and neuropeptide Y. Furthermore, changes in the degradation patterns of some of these neuropeptides, and also most of those derived from other larger proteins, did not follow specificity to proline. After a 4-day treatment, we found a significant amount of peptides, all derived from secreted pro-proteins, being cleaved with pair of basic residue specificity. In vitro experiments indicated that PREP modifies the endogenous dibasic residue specific proteolysis, in a KYP-2047 sensitive way. These findings suggest that PREP may act indirectly within the routes leading to the specific peptide changes that we observed. The data reported here suggest a wider tissue specific physiological role of PREP rather than the mere metabolism of proline containing active peptides and hormones.     

Specific reduction of carboxyl groups in peptides and proteins by diborane

1. The reaction of several peptides and proteins with diborane was studied under different conditions to determine those most suitable for the specific reduction of carboxyl groups. 2. In the reaction of model peptides and the cyclic peptides bacitracin and tyrocidin, reduction at 0 degrees was entirely specific for the carboxyl groups without affecting the peptide bonds. Acid amide residues were not reduced. Some tripeptides showed anomalous results in that the C-terminal residue was quite resistant to reduction. 3. Specific reduction of carboxyl groups was achieved in each of the following proteins: human serum albumin, egg albumin, adult human haemoglobin, sperm-whale apomyoglobin, horse heart cytochrome c and egg-white lysozyme. The C-terminal amino acid was usually reduced. 4. Conditions for specific reduction of all available carboxyl groups are not easily found and may vary from one substance to another. Specific reduction of a limited number of available carboxyl groups may be generally accomplished by reactions at -10 degrees . 5. It is suggested that this chemical modification, which has the advantage of permanence, may be useful in studying the role of carboxyl groups in the conformation of proteins and in the biological properties of peptides and proteins.     

The amino acid sequence of the alpha chain of the major haemoglobin of the rat (Rattus norvegicus).

1. A partial amino acid sequence of the alpha chain from the rat (Wistar, Rattus norvegicus) major haemoglobin is reported. The soluble tryptic peptides prepared from aminoethylated alpha-globin were separated by peptide 'mapping'. Sequencing of the tryptic peptides was carried out by the dansyl-Edman method and by the overlapping of smaller peptide fragments derived from secondary enzymic digestion. The insoluble 'core' peptides were further digested with chymotrypsin, thermolysin and pepsin to give smaller soluble peptides for sequencing. The tryptic peptides were ordered on the basis of their homology with the corresponding peptides of human alpha chain. 2. The proposed sequence is compared with that obtained by using an automated sequencer [Garrick et al. (1975) Biochem. J. 149, 245-258]. The differences in sequence resulting from the two methods are discussed. 3. It is suggested that the externally situated cysteine (residue 13) is responsible for the observed inhibition of crystallization of rat haemoglobin at alkaline pH. 4. Detailed evidence for the sequence has been deposited as Supplementary Publication SUP 50047 (9 pages) at the British Library (Linding Division), Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from which copies can be obtained on the terms given in Biochem. J. (1975) 145, 5.     

Structure-receptor binding relationships of sarafotoxin and endothelin in porcine cardiovascular tissues

The specific binding of 125I-sarafotoxin S6b was observed in the microsomal fractions from porcine thoracic aorta, and two vasoconstrictive peptides with strikingly homologous structures, sarafotoxin (SRT) and endothelin (ET), interact with a common receptor of the vasculature. The order of the potency of an each endothelin or sarafotoxin analogue as a competitor against 125I-sarafotoxin S6b binding was ET-1 greater than ET-2 greater than SRT S6b greater than ET-3 much greater than SRT S6c. The hydrophobic carboxyl-terminal tail and intramolecular disulfide bridges are essential for the binding activity. In addition, Ser4, Ser5 and Lys9 seem to be important for the activity while the 6th residue does not affect the activity.     

Screening of ACE-inhibitory peptides from a random peptide-displayed phage library using ACE-coupled liposomes

Angiotensin I converting enzyme (ACE)-inhibitory peptides were screened from a random peptide-displayed phage library using ACE-coupled liposomes. Among four kinds of inhibitory peptides selected by biopanning with two different elution strategies, a peptide (LSTLRSFCA) showed the highest inhibitory activity with an IC(50) value of 3microM. By measuring inhibitory activities of fragments of the peptide, it was found that the RSFCA region was a functional site to inhibit strongly the ACE catalytic activity, and particularly both Arg and Cys residues were essential for the strong inhibitory activity. The inhibitory activity of RRFCA was slightly increased, while that of the RSFRA, in which the Cys residue was replaced by Arg, was decreased to greater extent in comparison with the inhibitory activity of RSFCA. Taking into account the results obtained from the SPOT analysis, it was suggested that the Arg and Phe residues in RSFCA were important for a specific interaction with ACE, and the Cys residue inhibited the ACE activity. The cystein-based ACE-inhibitory peptides have not been isolated from processed food materials. These findings suggested that the biopanning method utilizing protein-coupled liposomes and random peptide libraries might have a possibility to screen new functional peptides that are not found in processed food materials.     

Expression of biologically active recombinant rat IgE-binding protein in Escherichia coli

IgE-binding protein (epsilon BP) is a protein which has affinity for IgE and was originally identified in rat basophilic leukemia (RBL) cells. Subsequently, it was found to be the rat homolog of CBP35, a murine beta-galactoside-specific lectin. This protein is also designated as L-34 and RL-29 and studied independently by several laboratories. More recently, CBP35 (epsilon BP) was found to be equivalent to Mac-2, a surface marker on activated macrophages. Using rat epsilon BP cDNA, we have succeeded in expressing recombinant epsilon BP in Escherichia coli. Milligram quantities of homogeneous epsilon BP could be obtained from bacterial lysate in a one-step affinity purification procedure utilizing lactosyl-Sepharose 4B and elution with a lactose gradient. The recombinant epsilon BP (r epsilon BP) binds mouse IgE and retains reactivity to anti-peptide antibodies specific for a sequence within rat epsilon BP. The purified r epsilon BP exhibits binding activity to various saccharides, with affinity for N-acetyllactosamine greater than thiodigalactoside greater than lactose much greater than D-galactose greater than L-arabinose, an order identical to that exhibited by native epsilon BP isolated from RBL cells. The recombinant lectin displayed hemagglutination activity when tested with rabbit erythrocytes. Although epsilon BP shares sequence homology to other lectins containing S-type (thiol-dependent) carbohydrate-recognition domains, r epsilon BP is resistant to air oxidation and does not require reducing agents for maintaining its activity. Furthermore, the single cysteine residue appears to be unexposed and can be alkylated only when the protein is denatured in 5.6 M guanidinium hydrochloride. The availability of a source for a large quantity of epsilon BP should facilitate the analysis of biological function(s) and structure-activity relationships of this lectin.     

Novel cyclic azo-bridged analogs of gonadotropin-releasing hormone.

Five linear analogs of GnRH containing a p-aminophenylalanine (Pap) residue in their sequence and their six corresponding azo-bridged cyclic derivatives were synthesized. The precyclic peptides were prepared on solid-support, while azo-cyclization was performed in solution by diazotization of the p-aminophenylalanine residue followed by intramolecular coupling of the formed diazo salt with either tyrosine or histidine side chains present in the sequence. All peptides were examined for their binding ability to the GnRH receptor expressed on rat pituitary membranes and for their LH-release activity from dispersed rat pituitary cells. Linear analogs 1 i.e [Pap(5)] GnRH and 3, i.e. [Tyr(3), Pap(5)] GnRH, were found to bind to the GnRH receptors only slightly less avidly than native GnRH. Their cyclization, however, led to a marked reduction in the binding capacity, i.e. from IC(50) of 10(-9) M to the 10(-7) M range, and in biopotency, i.e. LH-release. All other linear and cyclic peptides were found to bind selectively to the GnRH receptor only in the low microM range. Only peptide 1 was found comparable to native GnRH in respect to LH-release activity and thus may potentially be a good agonist of the parent peptide. Peptides 1-4, the most potent GnRH receptor binders, were examined for their conformational properties using CD. Cyclic-azo peptides 2 and 4 were further evaluated by NMR spectroscopy in solution combined with molecular modeling. The structural information obtained explains in part the GnRH-like biological activity observed.     

Construction and design of single stranded collagen-like structure

Polytheonamide B, a 48 residue long highly cytotoxic polypeptide extracted from marine sponges contains amino acids of alternate chirality and the N-terminal region is rich in t-Leu residues. The aim of this study is to analyze the effect of these alternate chiralities and conformational behavior of various model peptides containing t-Leu, in order to explore their role in designing bioactive peptides that shall offer advantages comparable to polytheonamide B, while circumventing its limitations. The conformational behavior of various peptides constructed from t-Leu of the form Ac-(L/D-X-L/D-Y)n-NHMe, where X = Gly/Ala/Leu and Y = t-Leu has been studied and compared with the corresponding peptides containing Leu residue. The results show that the helix driving capacity of L and D forms of t-Leu is less than that of Leu residue. In poly t-Leu peptides, the population of collagen/inverse collagen-type structures or right/left handed-helical structures for L and D forms respectively is found to be chain length-dependent. The stability of the helical structures is increased by -2 kcal per residue over the collagen-type structure in poly t-Leu peptides with chain length greater than five residues. Molecular view of peptides in collagen-type structure shows that the bulky side chains of t-Leu residues mask the NH moieties of the peptide bond, while the carbonyl groups lying along the helical groove are accessible to the small solvent molecules. Molecular model building suggests that one ethylene glycol molecule interacts by forming hydrogen bonds with carbonyl groups of two adjacent t-Leu residues. To the best of our knowledge, this is the first study of its own kind on the construction of a single-strand collagen/inverse collagen-type structure using unusual amino acid residues. Such synthetic collagen mimetic peptides shall exhibit specific affinity to natural collagen under controlled thermal conditions (heat or laser treatment) and hence can be explored as a new targeting method to attach therapeutic drugs to collagens in the living tissues and to biomaterials that incorporate natural collagens.     

Succinimide formation from aspartyl and asparaginyl peptides as a model for the spontaneous degradation of proteins

Nonenzymatic intramolecular reactions can result in the deamidation, isomerization, and racemization of protein and peptide asparaginyl and aspartyl residues via succinimide intermediates. To understand the sequence dependence of these reactions, we measured the rate of succinimide formation in a series of synthetic peptides at pH 7.4. These peptides (Val-Tyr-Pro-X-Y-Ala) contained an internal aspartyl, asparaginyl, aspartyl beta-methyl ester, or aspartyl alpha-methyl ester residue (X) followed by a glycyl, seryl, or alanyl residue (Y). The rates of succinimide formation of the asparaginyl peptides were found to be 13.1-35.6 times faster than those of the aspartyl peptides. The rates of succinimide formation for the glycyl peptides were 6.5-17.6 times faster than those of the alanyl peptides, while the rates for the seryl peptides were 1.6-4.5 times faster than those of the alanyl peptides. The overall 232-fold range in these reaction rates for aspartyl and asparaginyl residues suggests that sequence can be an important determinant in their stability in flexible peptides. In proteins, there may be a much larger range in the rates of succinimide formation because specific conformations may greatly enhance or inhibit this reaction.     

Identification of peptide hormones of the amphipathic helix class using the helical hydrophobic moment algorithm

Eisenberg's helical hydrophobic moment (less than mu H greater than) algorithm was applied to the analysis of the primary structure of amphipathic alpha-helical peptide hormones and an optimal method for identifying other peptides of this class determined. We quantitate and compare known amphipathic helical peptide hormones with a second group of peptides with proven nonamphipathic properties and determine the best method of distinguishing between them. The respective means of the maximum 11 residue less than mu H greater than for the amphipathic helical and control peptides were 0.46 (+/-/-0.07) and 0.33 (0.07) (P + 0.004). To better reflect the amphipathic potential of the entire peptide, the percent of 11 residue segments in each peptide above a particular less than mu H greater than was plotted vs less than mu H greater than. The resulting curves are referred to as HM-C. The mean HM-C (of the two groups) was highly significantly different such that the HM-C method was superior to others in its ability to distinguish amphipathic from nonamphipathic peptides. Several potential new members of this structural class were identified using this approach. Molecular modeling of a portion of one of these, prolactin inhibitory factor, reveals a strongly amphipathic alpha helix at residues 4-21. This computer-based method may enable rapid identification of peptides of the amphipathic alpha-helix class.     

Haemoglobins of the shark, Heterodontus portusjacksoni. III. Amino acid sequence of the beta-chain

The amino acid sequence of the beta-chain of the principal haemoglobin from the shark H. portusjacksoni has been determined. The chain has 141 residues, the same as that of mammalian alpha-chains and less than the 146 residues of mammalian beta-chains or the 148 residues of the alpha-chain from the tetrameric shark haemoglobin. The sequence was deduced from the sequences of peptides obtained by digestion of the globin or its cyanogen bromide fragments with trypsin, chymotrypsin, pepsin and papain. The difference in length of the beta-chain is most readily accounted for by the absence of the D helix. This small helical section is normally present in myoglobins and beta-globins but absent in alpha-chains. The deduction that it is absent from shark beta-chain is based on consideration of homology. The beta-chain shows the insertion of histidine beta2 and the deletions corresponding to residues A17 and AB1 relative to alpha-and myoglobin chains. The reactive thiol group in shark haemoglobin was shown by radioactive labelling to be residue 51 in the beta-chain, immediately preceding the E helix. The amino acid sequence of shark beta-chain shows 92 differences from human beta-chain, significantly more differences than shown by chicken or frog beta-chains, in line with its earlier time of divergence. If the tertiary structure of the shark beta-chain is the same as that of the horse then there are two changes in the alpha1beta2 contact site in oxyhaemoglobin and an additional one in deoxyhaemoglobin. When both alpha- and beta-chain contacts are considered there is a total of nine changes in residues involved in the alpha1beta2 contacts. There is no Bohr effect in shark haemoglobin, and of the residues normally involved in this effect the C-terminal histidine residue of the beta-chain is present, but the aspartyl (FG1) residue to which it is salt-linked is not, being replaced by a glutamyl residue.     

Specific cyanylation and cleavage at cysteine-104 human hemoglobin alpha-chain. A novel approach to the problem of the alpha-chain tryptic core in the study of haemoglobin variants by "fingerprinting" methods.

1. A new approach to the analysis, by "fingerprinting", of the tryptic core region of human haemoglobin alpha-chain is described. 2. The alpha-chain is cyanylated at its single cysteine residue (alpha104) and then split, by exposure to mild alkali, at the N-peptide bond of the resulting beta-thiocyanoalanine residue. 3. The two cleavage fragments, alpha1-103 and alpha104-141, are separated by gel filtration, and the fragment alpha104-141, which contains all the residues of the alpha-chain tryptic core, is digested with pepsin. 4. Preparative "fingerprints" of these peptic peptides yield eight major peptides, which provide complete sequence information for the whole region alpha104-141. 5. The utility of the method is demonstrated by repeating the determination of the substitution in haemoglobin Hopkins-2, a known alpha-chain core variant in which histidine-alpha112 (G19) is replaced by an aspartic acid residue.     

Amino acid requirement for the high affinity binding of a selected arginine-rich peptide with the HIV Rev-response element RNA.

The arginine-rich motif is a class of short arginine-rich peptides that bind to specific RNA structures that has been found to be a versatile framework for the design and selection of RNA-binding peptides. We previously identified novel peptides that bind to the Rev-response element (RRE) RNA of the HIV from an arginine-rich polypeptide library (ARPL) consisting of a polyarginine (15 mer) randomized at the N-terminal 10 positions. The selected peptides bound more strongly to the RRE than the natural binding partner, Rev, and contained glutamine residues that were assumed to be important for recognition of the G-A base pair. In addition, the peptides were predicted to bind to the RRE in an alpha-helical conformation. In this study, in order to understand the mechanism of the interaction between the RRE and the putative alpha-helical glutamine-containing peptides, the amino acid requirements for high affinity binding were analyzed by a combinatorial approach using a bacterial system for detecting RNA-peptide interactions. A consensus peptide, the DLA peptide, was elucidated, which consists of a single glutamine residue within a polyarginine context with the glutamine residue flanked at specific positions by three nonarginine residues, two of which appear to be important for alpha-helix stabilization. In addition, the DLA peptide was found to bind extremely tightly to the RRE with an affinity 50-fold higher than that of the Rev peptide as determined by a gel shift assay. A working model for the interaction of the DLA peptide to the RRE is proposed, which should aid in the development of peptide-based drugs that inhibit HIV replication, as well as in our understanding of polypeptide-RNA interactions. Copyright (c) 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis of enzymatically resistant nociceptin-related peptides containing a carbamic acid residue.

Nociceptin, a 17-amino acid peptide (FGGFTGARKSARKLANQ, N/OFQ), is the endogenous ligand of the nociceptin/orphanin FQ (NOP) receptor. This receptor-ligand system is involved in various physiological as well as pathophysiological mechanisms, but owing to the peptidic structure, it is rapidly degraded by enzymes. The enzymatic digestion of nociceptin involves mainly aminopeptidases and yields Noc(2-17)-OH and other smaller fragments. We aimed at increasing the enzymatic stability against aminopeptidases in the case of peptide Noc(1-13)-NH(2), which possesses the minimum sequence capable of interacting with the NOP receptor. Therefore we developed a new procedure for the synthesis of peptides with the carbamic acid residue [...-NH-CH(R)-CO-NH-CO-NH-CH(Q)-CO-.]. A set of four carbamic acid-nociceptin derivatives were produced. The carbamic acid residue was incorporated into the inner part of the peptides, building on solid phase, by using a suitable dipeptide fragment with carbamic acid residue produced by a simple and efficient three-step solution phase procedure. Enzymatic stability of carbamic acid peptides was studied in the presence of aminopeptidase M (AP-M) and in rat brain membrane homogenate. The receptor-binding properties were also studied by radioligand binding assay on crude rat brain membranes and the activity of the ligands were analyzed on isolated mouse vas deferens (MVD) tissues. We found that incorporation of the carbamic acid residue into the N-terminal part of nociceptin significantly increases the resistance against AP-M. We observed the decrease of binding affinities to the NOP receptor in case of the peptides modified in the N-terminal portion. Consequently, the incorporation of the carbamic acid residue into peptides can be proposed as a promising and reasonable tool for increasing enzymatic stability, where the native molecule is less sensitive for carbamic acid residue-related structural change.     

A cleavage cocktail for methionine-containing peptides.

A new cocktail has been developed for cleavage and deprotection of methionine-containing peptides synthesized by 9-fluorenylmethoxycarbonyl (Fmoc)-based solid-phase peptide synthesis methodology. The cocktail (trifluoroacetic acid 81%, phenol 5%, thioanisole 5%, 1,2-ethanedithiol 2.5%, water 3%, dimethylsulphide 2%, ammonium iodide 1.5% w/w) was designed to minimize methionine side-chain oxidation. Application of the new cocktail (Reagent H) is demonstrated with the synthesis of a model pentadecapeptide from the active site of DsbC, a periplasmic protein involved in protein disulphide bond formation. The model peptide, which contains one methionine and two cysteine residues, was cleaved with several cleavage cocktails, including Reagent H. The crude peptides obtained with the widely used cocktails K, R and B were found to be 15% to 55% in the methionine sulphoxide form, whereas no methionine sulphoxide was detected in the crude peptide obtained by cleavage and deprotection with Reagent H. Also, no methionine sulphoxide was detected when 1.5% w/w NH4I was added to cocktails K, R and B; however, the yield of the desired peptide was less than with Reagent H. A second 28 amino acid model peptide of the active site of DsbC was also cleaved and deprotected with Reagent H. The reduced dithiol form of the peptide was found to be the major component (51% yield) of the crude peptide obtained by cleavage for 3 h. When the cleavage time was extended to 10 h, the peptide was converted to the intramolecular disulphide form (35% yield). A proposed mechanism for the in situ oxidation of cysteine with Reagent H is presented.     

Characterization of Functional Receptors for Vasoactive Intestinal Peptide in Bovine Cerebral Arteries

This study reports the characterization of receptors for vasoactive intestinal peptide (VIP) on membranes prepared from bovine cerebral arteries. By use of HPLC we prepared two purified monoiodinated VIP radioligands with nearly equivalent cerebral vasorelaxant potency as native VIP, [Tyr(125I)10 )VIP and [Tyr(125I)22]VIP. The former resulted in a higher proportion of specific binding to arterial membranes than the latter and was therefore thought to be the superior radioligand for receptor characterization. The binding of [Tyr(125I)10]VIP to cerebral arterial membranes was saturable, specific, reversible, and dependent on time and temperature. Scatchard analysis suggested the presence of a high- and a low-affinity binding site with KD values of 0.2 and 11 nM and receptor concentrations of 79 and 737 fmol/mg of protein, respectively. The dose-response curves for binding to the VIP receptor by the VIP-homologous peptides PHI, PHM, and rat growth hormone-releasing factor (GRF) were very similar to their dose-response curves for relaxation of cerebral arteries. The order of potency was VIP greater than PHM greater than PHI greater than rat GRF. It is suggested that the characteristics of the vascular VIP binding sites and the close correlation between the binding and vasorelaxant properties of VIP and its related peptides argue for the vascular binding sites being functional receptors for VIP.     

Alternative processing pathways for preprovasoactive intestinal peptide in the enteric nervous system of the rat

In order to study biosynthetic processing of preprovasoactive intestinal peptide (prepro VIP) we have raised antisera to sequences that flank the biologically active peptides VIP and PHI (peptide with N-terminal His and C-terminal Ile). We have used these antisera in radioimmunoassays to identify the N-terminal flanking peptide (NFP) and C-terminal flanking peptide (CFP)-like immunoreactivities in rat brain and gastrointestinal tract. Concentrations of NFP-LI were similar to those of VIP in brain and throughout the gut. Concentrations of CFP-LI were 10-20% those of VIP-LI but could be increased 5-fold by digestion with carboxypeptidase B, suggesting that the C-terminal lysine residue of prepro VIP is not normally removed during processing. In rat stomach the NFP-LI was of higher molecular weight and greater hydrophobicity than the intestinal component. The data are consistent with alternative processing pathways for prepro VIP in enteric nerves of rat stomach and intestine.     

Sequence requirements for the activity of membrane-active peptides.

Synthetic peptides were constructed with the sequence of the first 20 residues of melittin and terminating with a range of different amino acid amides. These were found to have haemolytic and cytolytic activity similar to that of melittin, provided that certain charge constraints were observed. The nature of the 21st residue was not critical except when the residue introduced a negative charge. The presence of at least two positive charges in the molecule was found to be essential for activity. One of these charges could be the amino-terminal amine. Peptides could be inactivated by the addition of a non-acidic presequence which was acetylated at the N-terminus. Introducing a protease cleavable sequence into an N-terminal extension of the peptides produced analogues with low haemolytic activity that could be activated by proteolytic action. A peptide with extra positive charges introduced on the hydrophilic face of the helix possessed a haemolytic activity that was greater than that of melittin.     

Two new haemoglobins: haemoglobin Perspolis (alpha 64 (E13) Asp leads to Tyr) and haemoglobin J-Kurosh (alpha 19 (AB) Ala leads to Asp).

Two new haemoglobins are described which were found during a regular survey on voluntary blood donors in Iran. They are haemoglobin Perspolis [alpha 64 (E13) Asp leads to Tyr] and haemoglobin J-Kurosh [alpha 19 (AB) Ala leads to Asp]. The amino acid substitution in these two variants was determined by fingerprinting and amino acid analysis of the tryptic peptides and thermolytic peptides derived from abnormal tryptic peptides. Neither haemoglobin was associated with clinical symptoms.