John L. Morell

Synthetic magainin analogues with improved antimicrobial activity

Based on modifications to enhance the α‐helical structure of the broad spectrum antibiotic magainin 2, a series of analogues have been synthesized which display an increase up to two orders of magnitude in antimicrobial activity and, in the most favorable case, no appreciable increase in hemolytic activity over magainin 1 at the concentrations tested.

Properties and regulation of high-affinity pituitary receptors for corticotropin-releasing factor

Specific receptors for corticotropin-releasing factor (CRF) were identified in the rat anterior pituitary gland by binding studies with 125I-Tyr-CRF. Binding of the labeled CRF analog to pituitary particles was rapid and temperature-dependent, and reached steady state within 45 min at 22 degrees C. The CRF binding sites were saturable and of high affinity, with dissociation constant (Kd) of 0.76 X 10(-9) M. Pituitary binding of 125I-Tyr-CRF was inhibited by CRF, Tyr-CRF and the active 15-41 fragment of CRF, but not by the inactive 21-41 CRF fragment and unrelated peptides. The binding-inhibition potencies of the CRF peptides were similar to their activities as stimuli of adrenocorticotropic hormone (ACTH) release. The high-affinity CRF sites were markedly reduced in adrenalectomized rats, and this change was reversed by dexamethasone treatment. These data indicate that the high-affinity CRF sites demonstrated in the anterior pituitary are the functional receptors which mediate the stimulatory action of the peptide on ACTH release, and that CRF receptors are down-regulated during increased secretion of the hypothalamic hormone.

Magainin 2 amide and analogues Antimicrobial activity, membrane depolarization and susceptibility to proteolysis

We compared the abilities of synthetic magainin 2 amide and its analogues to inhibit the growth of Escherichia coli and to cause membrane depolarization in E. coli cells and cytochrome oxidase liposomes. The analogue, magainin A, was about 40‐times more active than magainin 2 amide in inhibiting the growth of E. coli and had a much more sustained effect on the membrane potential. In the liposomal system, however, there was only approx. 20% difference between these two peptides in the reduction of membrane potential and uncoupling of respiration. Studies with pronase digestion suggested that the difference in potency may be due to differential susceptibility to proteolysis in the presence of membranes.

The number and nature of α,β-unsaturated amino acids in nisin

Identical COOH-terminal sequences, viz. dehydroalanyllysine, have recently been established for the peptide antibiotics nisin [ 1 ] and subtilin [2] . The release of one equivalent of pyruvyllysine upon treatment of nisin at 100°C for 10 minutes with hydrogen chloride in glacial acetic acid [l] indicated the presence of one residue of the a&unsaturated amino acid. However, carboxymethylcysteine was found in excess of one residue in the hydrolysate of the addition product of mercaptoacetamide to nisin. The excess of carboxymethylcysteine over 1 residue did not result from &elimination of lanthionine. The test for sulfhydryl groups with maleimide was negative; the hydrolysate of the nisin-mercaptan addition product was free of cystine and contained the same number of lanthionine residues as nisin. Oxygen had to be excluded carefully in order to determine the presence of 2 residues of dehydroalanine and 1 residue of P-methyldehydroalanine in the antibiotic.

Agonist–antagonist properties of N-allyl-[D-Ala]2-Met-enkephalin

RECEPTOR displacement of the opiate antagonist naloxone requires concentrations of morphine and other opiate alkaloids similar to those required to elicit analgesia1–3 or inhibit ileal contraction4–5. A new class of opiates with structures based on the discovery of endogenous, morphine-like pentapeptide—enkephalin6—also displace opiate receptor binding with potencies paralleling in vivo activities7–10. Intrinsic activity (agonist or antagonist quality), a different dimension of opiate alkaloid effects in vivo, is closely correlated with the in vitro effects of sodium ion in the incubation medium: while antagonist binding to opiate receptors labelled by radioactive naloxone is almost unaffected by sodium ion addition, opiate alkaloid agonists become 10–60-fold weaker and mixed agonist–antagonists have intermediate downward shifts in apparent affinity after sodium ion addition1,11. We present here an analysis of the sodium sensitivities of the major opiate peptides which have all been identified as ‘agonists’ thus far. In addition, we describe the synthesis, behavioural and in vitro receptor analysis of N-allyl-[D-Ala]2-Met-enkephalin (Fig. 1), a novel peptide whose structure was designed in analogy12 with that of the alkaloid opiate antagonists13 in order to obtain a peptide opiate antagonist, if possible.

The reaction of cyanogen bromide with S-methylcysteine: fragmentation of the peptide 14-29 of bovine pancreatic ribonuclease A.

Summary The S-methylated peptide 14–29 of bovine pancreatic ribonuclease A is cleaved by cyanogen bromide with the formation of the expected fragments in 88% yield based on the conversion of S-methylcysteine.

Antibiotics and peptides with agonist and antagonist chemotactic activity

Abstract It has been found that the polypeptide antibiotics gramicidin S, tyrocidin and bacitracin, containing Leu-Phe or Ile-Phe sequences, are chemoattractants for neutrophils. Related synthetic pentapeptides containing the sequence Leu-Phe have stronger biological activities, provided the N-terminal residue is acylated. The formylated peptide f-L-Phe-D-Leu-L-Phe-D-Leu-L-Phe is a potent agonist (4 × 10 −9 M) whereas the t-butyloxycarbonyl analog is a good antagonist (8 × 10 −7 M).

Phosphorylation of magainin-2 by protein kinase C and inhibition of protein kinase C isozymes by a synthetic analogue of magainin-2-amide

Magainins are a family of antimicrobial peptides present in the skin extracts of Xenopus laevis. Both magainin‐1 and ‐2 do not have any significant effect on the activity of protein kinase C (PKC). Magainin‐2 was found to be readily phosphorylated by PKC to 0.5 mol‐32P/mol of peptide. Neither magainin‐1, which has a sequence of S8AGK and not S8AKK as in the case of magainin‐2, nor the magainin‐2 analogue with substitution of Ala for Ser8 was phosphorylated by the kinase, suggesting that Ser8 is the phosphorylation site of magainin‐2. One synthetic analogue of magainin, designated magainin B, which has a greater tendency for α‐helix formation in non‐aqueous environment than the parent peptide resulting from substitution of Ser8, Gly13, and Gly18 with Ala in magainin‐2‐amide, is a potent inhibitor of PKC. This peptide inhibits all three PKC isozymes with IC50 less than 20 μM. Magainin B also inhibits the binding of [3H]phorbol 12,13‐dibutyrate to the kinase. These results suggest that magainin‐2 may be modified by PKC through phosphorylation and that certain synthetic analogues of magainins may be used as inhibitors of PKC.

NONENZYMATIC CLEAVAGE OF PEPTIDE BONDS AND MULTIPLE-MOLECULAR FORMS OF ENZYMES

Nonenzymatic cleavage of peptide bonds is a relatively new technique for the fragmentation of peptides and proteins. As is implied by the term “nonenzymatic,” the commonly employed tools of peptide and protein fragmentation-the proteolytic enzymes-are no longer used. They are replaced by simple chemical compounds. Several of these reagents will be discussed later. Prerequisite for the nonenzymatic cleavage of a peptide bond is the presence of a functional group in the side chain of one of the two participating amino acids. Certain techniques for the nonenzymatic cleavage of peptide bonds are highly selective, and the reagent employed will react with one, and only one, of the commonly occurring amino acids. These techniques are applicable to protein fragmentation. Once applied to isozymes, they are capable of producing fragments which are microheterogeneous. I