Miklos Bodanszky

Unsulfated C-terminal 7-peptide of cholecystokinin: a new ligand of the opiate receptor.

Abstract On the basis of structural and conformational similarities between the C-terminal 7-peptide of cholecystokinin (pancreozymin) (CCK-(27–33)) and the active enkephalin analog [Trp 4 ,Met 5 ]-enkephalin, the affinity of CCK-(27–33) for the opiate receptor was determined. With unsulfated CCK-(27–33) half-maximal inhibition of stereospecific binding of [ 3 H]-naloxone in a rat brain membrane preparation was observed at a 200 times higher concentration than that required with [Met 5 ]-enkephalin. Sulfated CCK-(27–33) did not bind at concentrations up to 4 × 10 −5 M. In the bioassay based on inhibition of electrically evoked contractions of guinea pig ileum similar potency ratios were observed and the effect of CCK-(27–33) was shown to be naloxone-reversible. These findings are of interest in view of the recently demonstrated presence of CCK-fragments in the brain.

SIDE REACTIONS IN PEPTIDE SYNTHESIS

In two reviews the author, with J. Martinez, attempted a systematic discussion of side reactions encountered in peptide synthesis. The first of these articles [1] was organized according to the amino acids which cause or suffer changes, because the individuality of amino acids leads to side reactions which are characteristic for a particular residue. Of the twenty amino acid constituents of proteins only alanine and leucine are immune from characteristic side reactions, although not from racemization. The second, more comprehensive article [2] contains also a treatment of side reactions related to methods for protection and coupling. In this volume, in which an attempt is being made to discern the principles that govern peptide synthesis, side reactions proceeding through similar mechanisms are grouped together. Racemization will be treated among other undesired reactions.

A preferred conformation in the vasoactive intestinal peptide (VIP). Molecular architecture of gastrointestinal hormones

Abstract The ORD spectrum of the vasoactive intestinal peptide (VIP) in water indicates a preferred conformation with low helix content. Addition of organic solvents, especially of trifluoroethanol, results, even at low solvent concentration, in spectra with pronounced helical character. The readiness of shorter chains, with C-terminal sequences of VIP, to take up helical conformation under the effect of organic solvents parallels their biological activity. This suggests that an “active architecture” may be required for the interaction between hormone and receptor.

Contractile activity of vasotocin, oxytocin, and vasopressin on mammalian prostate.

The neurohypophyseal peptides arginine vasotocin, oxytocin and arginine vasopressin contracted guinea pig, rat, canine and human prostates with potencies and efficacies that were comparable to those of noradrenaline and methacholine. All three neuropeptides raised prostatic tone and elicited contractions at 10(-9) or 10(-8) M, with an order of efficacy: arginine vasotocin greater than oxytocin greater than arginine vasopressin. The findings suggest a physiological role for these peptides in prostatic smooth muscle contraction and possibly also in other aspects of male reproductive function.

STEPWISE SYNTHESIS OF PEPTIDES BY THE NITROPHENYL‐ESTER METHOD

When, about sixty years ago, the classic studies of Emil Fischerl proved that proteins are built up from amino acids, the synthetic methods devised by him and by Theodor Curtius2 were adequate enough to link amino acids to each other by peptide bonds. Thirty years later the introduction of the carbobemoxy protecting group discovered by Bergman and Zervass made it possible to synthesize simple peptides such as carnosin: anserin,6 and glutathion.6 Ten years ago a number of new methods were developed that could all be classified as mixed anhydride methods (within this group can be included also the socalled amino group activating methods). The discovery of the mixed anhydride procedure^^^-^ was most opportune. Almost simultaneously, structures of relatively complicated peptides like oxytocin and the vasopressins were elucidated by du Vigneaud and his co-workerss and, with the new methods, the synthesis of these hormones%-b could be achieved. More recently a number of interesting and important natural products of peptide structure have been synthesized, such as the antibiotic Gamicidin S,l0 the substances raising blood pressure, angiotensinsl’a-b, still later, aand &melanotropins.l%-c It was essentially the aforementioned synthetic methods that were applied in these syntheses. However the synchronization of progress in analysis and synthesis is no longer present in protein chemistry. Recent developments in the study of amino acid composition and sequence in proteins has led to the fascinating elucidation of structures as complex as insulin,1a ACTH,“ glucagon,ls and even of ribonuclease,’6 thereby far outstripping our synthetic capacity. The present state of synthesis seems to be unable to compete with the methods of analysis. Consequently not only the practical preparation of these hormones and enzymes is impossible at the moment, but even the proof of their structure by synthesis still lies in the future. Above a certain molecular size in a peptide, the difficulties of synthesis arise from several factors. There is the problem of the conformation of peptides, that is, the secondary structures due to such factors as hydrogen bonding, folding, and helix structure that may require special planning not known as yet. Another problem arises where, in a peptide to be synthesized, there is more than one disulfide bridge. The formation of a ring with an S S bridge by oxidation of two SH groups to a disulfideso brilliantly achieved by du Vigneaud and his co-workers17-is not practical if more than two SH groups are to be linked. In any event, apart from these special problems, the more general problem of peptide bond formation is only partially solved by the presently available methods that have been successfully applied in the synthesis of smaller molecules. Building a long chain involves many synthetic steps and, unless all of these steps can be performed with excellent yields, unusually large amounts of starting materials have to be used to obtain a decent quantity a t the comple-

Sepharose-avidin column for the binding of biotin or biotin-containing peptides

Avidin konnte an das Polysaccharid Sepharose 4B, das zuvor mit Bromcyan aktiviert wurde, gebunden werden. Um Biotin oder Biotin-haltige Peptide zu binden, erwies sich eine Sepharose-Avidin-Säule als besonders günstig.

Interaction of Peptides Related to Secretin with Hormone Receptors on Pancreatic Acinar Cells

Three analogues of the carboxyl-terminal tricosapeptide of secretin (S5-27), one with glutamine replacing glutamic acid in position 9 (9-Gln-S5-27), a second with asparagine substituted for aspartic acid in position 15 (15-Asn-S5-27), and a third with both replacements (9-Gln-15-Asn-S5-27) were tested for their ability to interact with hormone receptors on dispersed pancreatic acinar cells. Each of these analogues inhibited binding of 125I-labeled vasoactive intestinal peptide (VIP). None of the analogues increased cellular cyclic AMP but each inhibited the increase in cellular cyclic AMP produced by secretin or VIP. At the high affinity VIP receptor (the low affinity secretin receptor) each analogue had an apparent affinity which was greater than that for S5-27, whereas at he low affinity VIP receptor (the high affinity secretin receptor), each of the analogues had an apparent affinity which was the same as that for S5-27. Thus, in S5-27, substituting glutamine in position 9 or asparagine in position 15 makes the fragment more VIP-like but not less secretin-like. These results also provide additional evidence that the receptor having a low affinity for secretin and a high affinity for VIP is functionally distinct from the receptor having a high affinity for secretin and a low affinity for VIP.

Structure and synthesis of malformin A1

Abstract The structure of malformin A 1 , a metabolic product of Aspergillus niger , was reexamined and the sequence of its amino acid constituents established as The cyclopentapeptide-disulfide corresponding to this structure was prepared through stepwise synthesis of the protected pentapeptide derivative, benzyloxy-carbonyl- l -isoleucyl- S -benzyl- d -cysteinyl- S -benzyl- d -cysteinyl- l -valyl- d -leucine methylester, which in turn was converted to the hydrazide, partially deprotected, and cyclized via the azide. On removal of the S-benzyl groups and oxidation to the disulfide, a synthetic material was obtained that was indistinguishable from natural malformin A 1 and was as equally potent in causing curvatures on corn roots.

The importance of the amino acid in position 27 of cholecystokinin in determining its biological activity on pancreatic acini

We tested the synthetic C-terminal heptapeptide of cholecystokinin, which has the same biologic activity as cholecystokinin, and various synthetic analogs of the C-terminal heptapeptide for their abilities to increase amylase secretion from dispersed acini prepared from guinea-pig pancreas. We found that altering the chemical character of the amino acid in position 27 altered the potency with which the peptide stimulated amylase secretion but did not alter the efficacy of the peptide. We also found that, in the amino acid in position 27, the major function of the side-chain seems to be to position to the sulfate ester group at a proper distance from the backbone of the peptide chain, whereas the chemical structure of the side-chain per se seems to be of relatively minor importance.

Molecular architecture of peptide hormones optical rotatory dispersion of cholecystokinin-pancreozymin, bradykinin and 6-glycine bradykinin

Die optischen Rotationsspektren des intestinalen Hormons Cholecystokinin-Pancreozymin weisen, im Gegensatz zu denen des Secretins, auf das Fehlen bevorzugter Konformation und geben Einblick in den Zusammenhang zwischen molekularer Architektur und Hormonaktivität.