Biomedical subjects

R B Merrifield

Publications and source records attributed to R B Merrifield.

At least 37 records · Page 2Linked to original sources

PubMed1988

Binding and action of cecropin and cecropin analogues: antibacterial peptides from insects.

The mechanism of action of cecropin was studied by using liposomes as a model system. The bilayer was efficiently destroyed if the liposome net charge was zero or negative. Cecropin analogues with an impaired N-terminal helix had reduced membrane disrupting abilities that correlate with their lower antibacterial activity. The reduced bactericidal activity of the analogues was rationalized in terms of reduced binding to bacteria. The stoichiometry of cecropin killing of bacteria suggests that amounts of cecropin sufficient to form a monolayer strongly modify the bacterial membrane. Although some bacteria were resistant to cecropin they did bind large amounts in a non-productive manner. In contrast, mammalian erythrocytes achieve resistance by avoiding the binding of cecropin.

Animals↗

PubMed1988

Channel-forming properties of cecropins and related model compounds incorporated into planar lipid membranes.

Cecropins, positively charged antibacterial peptides found in the cecropia moth, and synthetic peptide analogs form large time-variant and voltage-dependent ion channels in planar lipid membranes in the physiological range of concentration. Single-channel conductances of up to 2.5 nS (in 0.1 M NaCl) were observed, which suggests a channel diameter of 4 nm. Channels formed by the peptides cecropin AD and MP3 had a permeability ratio of Cl-/Na+ = 2:1 in 0.1 M NaCl. A comparative study of the three cecropins, cecropins A, B, and D, and of six synthetic analogs allowed determination of structural requirements for pore formation. Shorter amphipathic peptides did not form channels, although they adsorbed to the bilayer. A flexible segment between the N-terminal amphipathic region and the C-terminal more hydrophobic region of the peptide was required for the observation of a time-variant, voltage-dependent conductance. Cecropin AD was the most effective voltage-dependent pore-forming peptide and was also the most potent antibacterial peptide against several test organisms. A positive surface charge or cholesterol in the bilayer reduced the conductances caused by cecropin AD or MP3 by at least 5-fold. This behavior is consistent with the known insensitivity of eukaryotic cells to cecropins. Our observations suggest that the broad antibacterial activity of cecropins is due to formation of large pores in bacterial cell membranes.

Amino Acid Sequence↗

PubMed1988

Potentiation of glucose-induced insulin release in islets by desHis1[Glu9]glucagon amide.

Glucagon and secretin and some of their hybrid analogs potentiate glucose-induced release of insulin from isolated mouse pancreatic islets. It was recently shown that the synthetic glucagon analog, desHis1[Glu9]glucagon amide, does not stimulate the formation of cyclic adenosine monophosphate in the rat hepatocyte membrane, but binds well to the glucagon receptor and is a good competitive antagonist of glucagon. In the present study the effect of this analog on isolated islets was examined. desHis1-[Glu9]glucagon amide at 3 x 10(-7) M, in the presence of 0.01 M D-glucose, increased the release of insulin by 30% and maintained that level for the full 30-min test period. The rate of insulin release returned to the glucose-induced base line after removal of the peptide. The same insulin level was produced by 3 x 10(-9) M glucagon, and at 3 x 10(-7) M glucagon insulin release was enhanced 290% above the glucose base line.

Amino Acid Sequence↗

PubMed1988

Insulin release by glucagon and secretin: studies with secretin-glucagon hybrids.

Secretin and glucagon potentiate glucose-induced insulin release. We have compared the effects of secretin and glucagon with that of four hybrid molecules of the two hormones on insulin release and formation of cyclic AMP (cAMP) in isolated mouse pancreatic islets. All six peptides potentiated the release of insulin at 10 mM D-glucose, and their effects were indistinguishable with respect to the dynamics of release, dose-response relationship, and glucose dependency. However, measurements of cAMP accumulation in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (10(-4) M) showed that the fold increase compared with glucose alone had the following ranking order: secretin = [Tyr10, Tyr13]-secretin 1.6 less than [Tyr10, Tyr13, Trp25]secretin 1.8 less than glucagon 1.9 less than [Asp3, Glu9, Arg12]glucagon 2.3 = [Asp3, Glu9]glucagon. These results suggest that despite similar potentiating effects of secretin and glucagon on glucose-induced insulin release, their modes of action may be different.

Amino Acid Sequence↗

PubMed1987

The relation of predicted structure to observed conformation and activity of glucagon analogs containing replacements at positions 19, 22, and 23.

Six new analogs of glucagon have been synthesized containing replacements at positions 19, 22, and 23. They were designed to study the correlation between predicted conformation in the 19-27 segment of the hormone and the conformation calculated from circular dichroism measurements and the observed activation of adenylate cyclase in the liver membrane. The analogs were [Val19]glucagon, [Val22]glucagon, [Glu23]glucagon, [Val19,Glu23]glucagon, [Glu22,Glu23]glucagon, and [Ala22,Ala23]glucagon. The structures predicted for the 19-27 segment ranged from strongly alpha helical to weakly beta sheet. The observed conformations varied as functions of amino acid composition, solvent, concentration, pH, and temperature but did not correlate well with prediction. There was, however, a correlation between predicted structure and activation of adenylate cyclase in rat liver membranes.

Adenylyl Cyclases↗

PubMed1987

Synthetic peptides VH(27-68) and VH(16-68) of the myeloma immunoglobulin M603 heavy chain and their association with the natural light chain to form an antigen binding site.

A 53-residue peptide corresponding to the variable region 16-68 of the heavy chain of phosphocholine binding mouse myeloma M603 protein was synthesized by a solid-phase fragment strategy. The homogeneity of the VH(16-68) peptide was confirmed by high-performance liquid chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, amino acid analysis, and mass spectrometry. Synthetic VH(16-68) associated with the M603 light chain, and about 27% of the recombination mixture bound to phosphocholine immobilized on Sepharose as compared to a 28% binding yield obtained for the recombined natural light and heavy chains under the same conditions. The binding yield for the recombinant of the light chain with previously prepared VH(27-68) fragment was about 11%. These semisynthetic antibodies VH(27-68) and VH(16-68) light chain recombinants are forerunners of structural variants designed to study the antigen binding pocket of the M603 immunoglobulin.

Amino Acid Sequence↗

PubMed1987

Glucagon antagonists. Synthesis and inhibitory properties of Asp3-containing glucagon analogs.

In an effort to find analogs of glucagon that would bind to the glucagon receptor of the rat liver membrane but would not activate membrane-bound adenyl cyclase, several hybrid molecules were synthesized which contained sequences from both glucagon and secretin. [Asp3, Glu9]Glucagon and [Asp3, Glu9, Arg12]glucagon were inactive in the adenyl cyclase assay even at high concentrations but retained some binding affinity for the receptor. They were able to displace 125I-glucagon completely from its receptor and could completely inhibit the activation of adenyl cyclase by natural or synthetic glucagon. The inhibition index [I/A]50 was approximately 110 for both analogs. [Asp3]Glucagon, [Glu3]glucagon and [Asp3, Lys17, 18, Glu21]glucagon were weak partial agonists, while [Asp3, Glu21]glucagon was inactive and a poor inhibitor. The peptides were synthesized by solid-phase methods and purified to homogeneity by reverse-phase high-performance liquid chromatography on C18 silica columns. These are the first fully synthetic competitive glucagon antagonists to be reported.

Adenylyl Cyclases↗

PubMed1987

Secretin stimulates cyclic AMP and inositol trisphosphate production in rat pancreatic acinar tissue by two fully independent mechanisms.

In rat pancreatic acinar tissue adenylate cyclase is stimulated by low concentrations of secretin, while higher concentrations also activate phosphatidylinositol bisphosphate hydrolysis. By the use of the secretin analogues [Tyr10,13]secretin and [Tyr10,13,Phe22,Trp25]secretin, we have shown that substitution of tyrosine for leucine at positions 10 and 13 was sufficient to reduce the ability of the peptide to stimulate the production of inositol trisphosphate and the increases in cytosolic free calcium, while the ability to stimulate cAMP is little affected and the peptide remained a full agonist. Incubation with cholera toxin caused increases in cAMP, which were maximal after 30 min. Cholera toxin treatment also resulted in a marked reduction of secretin-stimulated inositol trisphosphate production, but this required a much more prolonged treatment (150-240 min), suggesting that different cholera toxin substrates were involved. Activation of protein kinase C with the phorbol ester phorbol 12-myristate 13-acetate had no effect on secretin-induced cAMP formation, nor was secretin-stimulated inositol trisphosphate formation altered by further increases in cAMP. These results indicate that the mechanisms by which secretin stimulates adenylate cyclase and activates phospholipase C in acinar tissue are completely independent.

1-Methyl-3-isobutylxanthine↗

PubMed1987

Synthetic peptide antagonists of glucagon.

Several glucagon analogs were synthesized in an effort to find derivatives that would bind with high affinity to the glucagon receptor of rat liver membranes but would not activate membrane-bound adenylate cyclase and, therefore, would serve as antagonists of the hormone. Measurements on a series of glucagon/secretin hybrids indicated that replacement of Asp9 in glucagon by Glu9, found in secretin, was the important sequence difference in the N terminus of the two hormones. Further deletion of His1 and introduction of a C-terminal amide resulted in des-His1-[Glu9]glucagon amide, which had a 40% binding affinity relative to that of native glucagon but caused no detectable adenylate cyclase activation in the rat liver membrane. This antagonist completely inhibited the effect of a concentration of glucagon that alone gave a full agonist response. It had an inhibition index of 12. The pA2 was 7.2. An attempt was made to relate conformation with receptor binding. The peptides were synthesized by solid-phase methods and purified to homogeneity by reverse-phase high-performance liquid chromatography on C18-silica columns.

Adenylyl Cyclases↗

PubMed1987

4-Methoxybenzyloxycarbonyl amino acids in solid phase peptide synthesis.

Moz-amino acids were used in solid-phase synthesis of Leu-Ala-Gly-Val and thymosin alpha 1. It was found that the Moz-group can be removed rapidly and completely with 5-10% TFA in CH2Cl2. Some advantages of utilizing Moz-amino acids over Boc-amino acids in solid phase peptide synthesis are demonstrated.

Chromatography, High Pressure Liquid↗

PubMed1987

Synthesis and hormonal activity of [Tyr22] glucagon and [desHis1, Tyr22] glucagon.

[Tyr22] glucagon and [desHis1, Tyr22] glucagon were synthesized by an improved solid phase procedure on a Pam-resin. The course of the synthesis was monitored by quantitative ninhydrin analysis and preview sequencing. Following cleavage by the low/high HF method the peptides were purified by ion exchange chromatography and reverse phase HPLC. The overall yield of homogeneous isolated peptide from the first amino acid was 41%. Circular dichroism measurements on dilute solutions in mixed aqueous organic solvents at pH 2, 6.9 and 9.2 showed increased beta-sheet structure relative to glucagon. [Tyr22] glucagon was a full agonist with 20-30% activity in the rabbit blood glucose assay and 10% activity in the rat liver membrane adenyl cyclase assay. [desHis1, Tyr22] glucagon had only a trace of activity in the adenyl cyclase assay (less than 0.002%) but bound to membranes in a competitive [125I] glucagon assay 1.0% as well as glucagon. The analog completely inhibited formation of cAMP by natural glucagon, with 50% inhibition at a ratio of 83:1 and pA2 = 6.7. The data are discussed in terms of models of glucagon structure in dilute solution.

Adenylyl Cyclases↗

PubMed1986

A synthetic approach to structure-function relationships in the murine epidermal growth factor molecule.

Murine epidermal growth factor, a 53-amino acid peptide that is mitogenic for a number of cell types, has been synthesized by the solid-phase method. The synthetic peptide is identical to the natural material in amino acid composition, chromatographic behavior, receptor binding, and stimulation of DNA synthesis. Fragments of the EGF molecule corresponding to residues 42-53, 32-53, and 15-53 were constructed as well as the methionine sulfoxide derivative of EGF, [Met(O)21]EGF-(1-53), and a polymeric form of EGF. [Met(O)21]EGF-(1-53) was slightly less active than EGF in receptor binding and stimulation of DNA synthesis. Polymeric EGF was 1/100th as active as EGF, while EGF-(15-53) was less potent than EGF-(1-53) by a factor of 10(4). EGF-(32-53) was even less active and EGF-(43-53) was inactive.

Animals↗

PubMed1986

Solid phase synthesis of thymosin beta 9.

Thymosin beta 9, a 41 residue thymic polypeptide, has been synthesized by a solid phase method. A modification of the low HF method was used to deprotect and cleave the peptide from the resin. Thymosin beta 9 was then obtained in analytically pure form by a one-step purification procedure in 32% yield. The activity of thymosin beta 9 in the terminal deoxynucleotidyl transferase assay was greater than calf thymus fraction 5, but comparable to thymosin beta 4. In contrast to thymosin alpha 1, neither beta 4 nor beta 9 was active in the rosette inhibition assay.

Animals↗

PubMed1985

Solid-phase synthesis of PYLa and isolation of its natural counterpart, PGLa [PYLa-(4-24)] from skin secretion of Xenopus laevis.

From the nucleotide sequence of clones isolated from a cDNA library constructed from skin of Xenopus laevis, the existence of PYLa, a peptide comprised of 24 amino acids, was predicted. This peptide was synthesized by solid-phase methods and purified to homogeneity with an overall yield of 61%. The synthetic peptide was used as reference substance to search for its natural counterpart in skin secretion of Xenopus. Two peptides were found which were very similar to PYLa except for the absence of the first three amino acids. These 21-amino-acid peptides, termed PGLa, can be generated from PYLa by cleavage after the single arginine residue present in the latter. The two forms of PGLa differ in their retention time on HPLC but have identical amino acid compositions and terminal sequences. Tryptic hydrolysis of synthetic PYLa after the single arginine yields exclusively PGLa with the shorter retention time on HPLC. The chemical difference between the two forms of PGLa is currently not known. The possible biological role of these newly discovered constituents of frog skin secretion is discussed.

Amino Acids↗

PubMed1985

N-terminal analogues of cecropin A: synthesis, antibacterial activity, and conformational properties.

Six analogues of the 37-residue antibacterial peptide cecropin A were synthesized by the solid-phase method: cecropin A-(2-37), [Glu2]cecropin A, [Pro4]cecropin A, [Glu6]cecropin A, [Leu6]cecropin A, and [Pro8]cecropin A. Their antibacterial activities against four test organisms were determined and related to conformational changes observed in their CD spectra and were discussed on the basis of a previously proposed amphipathic alpha-helix model. An aromatic residue in position 2 was shown to be important for activity against all tested bacteria. The highly alpha-helical 1-11 region of cecropin A did not appear to play a significant role in its activity against Escherichia coli but was clearly involved in its interaction against Pseudomonas aeruginosa, Bacillus megaterium, and Micrococcus luteus.

Anti-Bacterial Agents↗

PubMed1985

On the primary structures of lysozyme, cecropins and attacins from Hyalophora cecropia.

Diapausing pupae of Cecropia respond to a bacterial infection by the selective synthesis of RNA and 15-20 hemolymph proteins. Of these we have purified lysozyme and two classes of antibacterial proteins called cecropins and attacins. The primary structure has been determined for the lysozyme, one attacin and five cecropins. We have also prepared a cDNA bank, isolated and sequenced clones corresponding to the lysozyme, the two main attacins and one cecropin. The results of these structural studies are briefly summarized. Finally we review the solid phase synthesis of cecropin A and B and 9 analogs of cecropin A.

Amino Acid Sequence↗

PubMed1985

Molecular cloning, cDNA sequencing, and chemical synthesis of cecropin B from Hyalophora cecropia.

Two cDNA clones containing coding information for cecropin B from the Cecropia moth (Hyalophora cecropia) were identified by means of a synthetic probe. Sequencing of the two inserts showed that cecropin B is processed from a 62-amino acid residue precursor molecule including a 26-residue leader peptide and a COOH-terminal glycine residue. The latter presumably donates the nitrogen of the amide group present on the COOH-terminal leucine residue of the mature cecropin B. The sequence deduced for the mature cecropin B differed in the COOH-terminal region from the tentative structure previously determined by carboxypeptidase digestion. To settle the discrepancy, cecropin B was synthesized according to the cDNA sequence with an amidated COOH-terminal leucine. Natural and synthetic cecropin B were found to be indistinguishable with respect to electrophoretic mobility and antibacterial activity against seven different bacteria. The COOH-terminal tetrapeptides were isolated from both natural and synthetic cecropin B and found to be indistinguishable. The correct sequence for cecropin B is (formula; see text).

Amino Acid Sequence↗

PubMed1985

Solid phase synthesis of gastrin I. Comparison of methods utilizing strong acid for deprotection and cleavage.

A successful synthesis of human gastrin I in 60% overall yield based on the first residue attached to a benzhydrylamine-resin was achieved by the stepwise solidphase method. The synthesis was carried out on a 1% crosslinked polystyrene support, using conventional benzyl-based side chain protecting groups and final deprotection with different acidic protocols. Several improvements in this general approach were applied, including new scavengers, new resin attachment and especially a new technique that allows the strong acid reactions to occur by an SN2 mechanism.

Amino Acids↗