Medium-sized Peptides Research Articles

Slow isomerization of some proline-containing peptides inducing peak splitting during reversed-phase high-performance liquid chromatography

A slow conformational equilibrium, commensurable with the retention times, was shown to induce peak broadening or peak splitting during reversed-phase high-performance liquid chromatography of several medium-sized peptides. Elution at 50°C resulted in sharp unique peaks, while at sub-ambient temperature well resolved peaks were observed. Linear peptides which show this phenomenon had a Pro-Pro bond, but the phenomenon was also observed in the case of a cyclic peptide containing two non-vicinal proline residues.

Humoral factors released during trauma of Aplysia body wall. II. Effects of possible mediators.

1. Preliminary, general chemical characteristics of substances in artificial sea water (ASW) washed through stimulated body wall (SBW) and in hemolymph taken from noxiously stimulated animals (SHL) were consistent with those of classical neurotransmitters, amino acids, and small- to medium-sized peptides. 2. 5-Hydroxytryptamine (5HT) and acetylcholine (ACh), unlike SBW and SHL, caused relaxation when perfused into isolated body wall. FMRFamide produced a biphasic response--brief contraction followed by prolonged relaxation. 3. Small cardioactive peptide (SCPB) caused body wall contractions similar to those produced by SBW and SHL, except that SCPB contractions displayed more desensitization and were completely blocked by 30 mM CoCl2. SCPB and SBW contractions were synergistic. 4. Dopamine caused persistent body wall contractions similar to those of SBW and SHL. Dopamine contractions were reduced but not blocked by 30 mM CoCl2. Unlike SBW activity, dopamine activity was reduced by alkalinization. 5. Glutamate and taurine produced strong but usually short-lasting body wall contractions. Adenosine, octopamine, arginine vasotocin, and cholecystokinin (CCK-8) caused weak or variable contractions. Met-enkephalin and somatostatin caused no obvious body wall responses. 6. When superfused over the fully sheathed abdominal ganglion, FMRFamide, met-enkephalin, glutamate, aspartate, and taurine reduced the magnitude of the gill-withdrawal reflex elicited by siphon nerve stimulation. 7. Taken together with earlier results, these data suggest a preliminary framework for trauma signal pathways. It is proposed that stress hormones (perhaps including FMRFamide, SCPs, 5HT, and dopamine) are released into hemolymph from neuroendocrine cells. Effective amounts of active intracellular solutes such as amino acids may also be released by extensive cellular rupture. Various humoral signals produce slow effects that contribute to hemostasis, balling up, increased cardiac output, and reflex suppression.

Design of Peptides and Proteins

This chapter focuses on contemporary approaches to the design of structurally defined peptides and proteins with special emphasis being given to the ways in which this approach has elucidated the structural basis for the function of a variety of natural molecules. When in their proper biological milieu, peptides and proteins assume fairly well-defined conformations, which are responsible for their biological and physical properties. The mechanisms by which these molecules adopt defined three-dimensional structures depend on their size and chemical compositions. There are a variety of strategies and approaches for protein and peptide design. Those adopted for a given problem will depend on the size and molecular characteristics of the desired target molecule. If one wishes to create a model for a small peptide (e.g., the pentapeptide hormone enkephalin), then it might be feasible to design an analog of this molecule that is locked into a biologically active conformation. The design of proteins that adopt predetermined three-dimensional structures is perhaps the most ambitious goal, and one that has been attacked only recently. The design of conformationally constrained peptides is a powerful method for elucidating the biologically active conformation of a peptide when it is bound to its receptor. The design of medium-sized peptides is beset with many of the same problems encountered in the design of small peptides. Medium-sized peptides are also very flexible and tend to lack defined conformations at room temperature in aqueous solutions.

Atrial Peptide Study Proceeds Apace

"THE HEART SEEMS to be a very self-serving, self-centered organ, and it seems to have devised a way to protect itself against the volume overload of high blood pressure." So said Tadashi Inagami, PhD, professor of biochemistry and director of the Specialized Center for Research on Hypertension at Vanderbilt University Medical Center, Nashville, Tenn, at the annual meeting of the American Heart Association (AHA) in Dallas. The "way" Inagami referred to is atrial natriuretic factor. How this medium-size peptide secreted by the atria (and probably, it is now thought, to some degree by the ventricles as well) does its job, why it doesn't always do it adequately, and how its influence can be augmented are topics of intense interest and speculation among cardiovascular specialists. Pharmaceutical companies also are seeking to learn more about the material whose properties make Marc Cantin, MD, director of the Laboratory of Experimental Hypertension at the

Quantification of chymosin action on nonlabeled κ-casein-related peptide substrates by ultraviolet spectrophotometry: Description of kinetics by the analysis of progress curves

A method is described for quantifying the proteolytic action of the milk-clotting enzyme chymosin on small and medium-sized peptide substrates by monitoring the decrease of absorbance at 230 nm during cleavage. The method is illustrated by the determination of the kinetic parameters of the specific splitting of a κ-casein-related hexa- and pentadecapeptide by chymosin. The results are in good agreement with those found earlier with the same enzyme/substrate system by using an automated ninhydrin method. Erroneous results were obtained when the kinetic data were derived from one single progress curve. The significance of initial rate measurements for calculating correct kinetic parameters is briefly discussed. The usefulness of single progress curves measured at different initial substrate concentrations for obtaining information about the mechanism of the enzymic reaction is demonstrated.

The effects of various peptides on the thermotropic properties of phosphatidylcholine bilayers.

The effects of an amino acid derivative (N-benzoyl-L-argininamide), four small peptides (Phe-Gly-Phe-Gly, gastrin-related peptide (Trp-Met-Arg-Phe-NH2), tetragastrin (Trp-Met-Asp-Phe-NH2), pentagastrin (Boc-beta Ala-Trp-Met-Asp-Phe-NH2] and one medium-sized peptide, glucagon (29 residues), on the gel-to-liquid crystalline transition of a multilamellar suspension of dimyristoylphosphatidylcholine have been studied by means of high-sensitivity differential scanning calorimetry. At low concentrations of added solutes, the temperature at which the excess apparent specific heat in the gel-to-liquid crystalline phase transition of the lipid is maximal is lowered by an amount proportional to the total concentration of the peptide, with proportionality constants ranging from -0.018 K mM-1 for Phe-Gly-Phe-Gly to -3.1 K mM-1 for the gastrin-related peptide. The lipid mixtures involving the first two solutes listed above exhibited approximately symmetrical curves of excess apparent specific heat vs. temperature. The curves for the other solutes were asymmetric, and could be well represented as the sum of either two or three two-state curves. The asymmetry, which was especially pronounced in the cases of pentagastrin and glucagon, thus appeared to be due to the presence of components having lower and/or higher transition temperatures than that of the lipid. Pentagastrin and glucagon (R.M. Epand and J.M. Sturtevant, Biochemistry 20 (1981) 4603) have much smaller effects on the gel-to-liquid crystalline phase transition of dipalmitoylphosphatidylcholine than on that of the dimyristoyl analog.

Simple, efficient ternary solvent system for the separation of luteinizing hormone-releasing hormone and enkephalins by reversed-phase high-performance liquid chromatography

Reversed-phase high-performance liquid chromatography based on hydrophobic interaction of amino acid side-chains with octyl-, octadecyl-, cyanopropyl-, or phenyl-silica-bonded stationary phases has become the method of choice for the purification and analysis of small- and medium-size peptides. In the ion-pair mode, the organic solvent modifiers commonly used are either acetonitrile or methanol in the presence of suitable counterions. Recently, instruments with ternary, and even quaternary solvent delivery systems have become available. Although these instruments are undoubtedly more powerful and versatile, they are also more complex to handle. Moreover, their high price is a deterrent for many laboratories. In this paper it will be demonstrated that when methanol and acetonitrile are used simultaneously as organic modifying agents, in many cases better separations can be obtained than when using a binary gradient with either acetonitrile or methanol alone. Briefly, gradient elution was used with methanol—water (25:75), containing linearly increasing amounts of acetonitrile—water (80:20) at constant 0.1% trifluoroacetic acid concentration. Five commercially available prepacked columns were compared, namely: Whatman Partisil ODS-3, Knauer LiChrosorb RP-8, Varian MicroPak MCH-5, Waters μBondapak C18, and Vydac 218TP. As reference peptides were used Met-Enk, Leu-Enk, Leu-Enk-Arg-Lys, Leu-Enk-Arg-Arg, dynorphin1–13, β-melanotropin, Lys-bradykinin, neurotensin, angiotensin, and luteinizing hormone-releasing hormone (LHRH). This simple, yet efficient gradient system was successfully applied to the separation and purification of de novo biosynthesized enkephalins and LHRH in trophoblastic shells of the human placenta.

Secondary structures of proteins and peptides in amphiphilic environments. (A review).

Many peptides and proteins that act at lipid--water interfaces assume a unique amphiphilic secondary structure which is induced by the anisotropy of the interface. By using synthetic peptides in which these inducible amphiphilic structures have been optimized, one can show that the amphiphilic alpha helix is a functional determinant of representative apolipoproteins, peptide toxins, and peptide hormones. By increasing the amphiphilicity of the structurally important regions of the molecule, one can enhance the biological activity of the peptide even beyond that of the naturally occurring polypeptide. It is proposed that rigid amphiphilic secondary structures such as alpha helix, beta sheet, or pi helix will be found in most medium-sized peptides acting at membranes and lipid--water interfaces.

Medium-sized peptides in the blood of patients with uremia.

We have carried out high performance liquid chromatographic analysis of serum and ultrafiltrate of blood obtained from uremic patients and normal subjects to elucidate the presence of medium-sized peptides unique to uremia. Many fluorescamine-positive substances, excluding amino acids and guanidine compounds, were increased in uremic serum compared with normal serum. At the 0.5-1.0 pmol/ml serum level, several peaks were unique to uremia. The retention time, fluorescamine reactivity, molecular weight distribution and the result of enzymatic digestion revealed that these peaks are peptidic substances.

Profiling of urinary medium-sized peptides in normal and uremic urine by high-performance liquid chromatography

This report describes the profiling of medium-sized peptides in both normal and uremic urine by ion-pair reversed-phase high-performance liquid chromatography using an acetonitrile—heptafluorobutyric acid solvent system as eluent. Several medium-sized peptide peaks could be detected in both normal and uremic urine at low picomole level by using post-column fluorescence derivatization with fluorescamine. Contrary to expectation, uremic urine contained slightly larger amounts of medium-sized peptides compared with normal urine.

A general procedure for the manual sequencing of small quantities of peptides

Modifications of the classical Edman cycle permitted the complete sequential degradation, without prior derivatization, of all small and medium-sized peptides with a free N-terminus. Sequences of up to 40 residues appeared possible with most long peptides. Quantities of 5–50 nmoles were optimal for the apparatus described. Cycling time was 50–90 min. Degradation was monitored and derivatives identified with a fast and sensitive thin-layer technique.

Comparison of the rates of proteolysis during ripening of Cheddar cheeses made with calf rennet and swine pepsin as coagulants

SummaryThe rates of proteolysis during ripening were followed in cheeses made with either calf rennet or swine pepsin and either starter or δ-gluconic acid lactone (GAL) as a replacement for the starter. A gel-filtration column technique and starch-gel electrophoresis were used for analysis, and bacterial counts were made on all samples. Proteolysis was faster in cheeses made using GAL than in those made using starter and also slightly faster in GAL cheeses made with swine pepsin than in those made with rennet. Further, it was considerably slower in starter-containing cheeses made with swine pepsin than in those made with rennet. It is suggested that these differences were due to the much greater rate of development of acidity in cheeses made with GAL than in those made with starter, which resulted in more of the coagulant being incorporated into the curd in an active state. The rate of proteolysis in starter-containing cheeses appeared to follow a characteristic course, being initially slow, then markedly increasing with a later slow decline. It is suggested that the increase in the rate of proteolysis was due to an increase in the total activity of bacterial proteinases released by lysis of the bacteria. Indications were obtained that the coagulants and bacterial proteinases catalysed broadly similar patterns of protein breakdown in cheese, and that medium-sized peptides (mol. wt 9000–14000) were formed as definite intermediates in the process. The results also showed that rennet and swine pepsin remained active for at least 7 months in GAL cheeses, that rennet contributed significantly to proteolysis in starter-containing cheeses, and that swine pepsin was at least extensively inactivated and possibly completely inactivated during cheese-making with starter.