Conformational stability of human erythrocyte transglutaminase. Patterns of thermal unfolding at acid and alkaline pH.

Tissue-type transglutaminase is irreversibly inactivated during heat treatment. The rate of inactivation is low at pH 7.5; it increases slightly at acid pH (6.1) but much more at alkaline pH (9.0-9.5), suggesting that specific effects take place in the alkaline range, possibly in relation to decreased stability of the transition-state intermediate as pH is raised above 9.0. Differential scanning calorimetry experiments indicate that thermal unfolding of the protein occurs with two separate transitions, involving independent regions of the enzyme. They are assigned to domains 1 and 2 and domains 3 and 4, respectively, by a combination of calorimetric and spectroscopic techniques. When considering the effects of pH, we noted that transglutaminase was unfolded via different pathways at the different pH values considered. At acid pH, the whole structure of the protein was lost irreversibly, with massive aggregation. At neutral and, even more so, at alkaline pH, aggregation was absent (or very limited at high protein concentration) and the loss of secondary structure was dependent on the ionization state of crucial lysine residues. Unfolding at pH 9.5 apparently chiefly involved the N-terminal region, as testified by changes in protein intrinsic fluorescence. In addition, the C-terminal region was destabilized at each pH value tested during thermal unfolding, as shown by digestion with V8 proteinase, which is inactive on the native protein. Evidence was obtained that the N-terminal and C-terminal regions interact with each other in determining the structure of the native protein.

Conformational aspects of human formylpeptide receptor antagonists.

The conformation of several Phe-D-Leu-Phe-D-Leu-Phe analogues was analyzed using infrared absorption and circular dichroism. Their effect on human neutrophils was verified by receptor binding and chemotaxis assays. The results demonstrate that the compounds examined prefer an ordered conformation (beta-turn) in amphipatic environment, and that they are able to antagonize the neutrophil functions evoked by CHO-Met-Leu-Phe.

Synthesis, biological activity and conformational studies of geometrically restricted tripeptide analogues of the chemoattractant HCO-Met-Leu-Phe-OMe.

The formyl tripeptides containing 2-azetidinecarboxylic acid 2, 2-piperidinecarboxylic acid 3 and norvaline 4 in position 2 were synthesized and their biological activity was evaluated. The conformation of peptides was studied by CD and FT-IR techniques. While 2 and 3 do not show either chemotactic activity or superoxide production, 4 retains both activities.

The importance of the peptide bond at position 2 in HCO-Met-Leu-Phe-OMe analogues as shown by studies on human neutrophils.

The formylpeptides formyl-methionyl-N-methylleucyl-phenylalanine methyl ester [for-Met-(NMe)Leu-Phe-OMe] 1, formyl-methionyl-2-aminotetralin-2-carboxyl-phenylalanine methyl ester [for-Met-Atc-Phe-OMe] 2, formyl-methionyl-1,2,3,4-tetrahydroisoquinoline-3-carboxyl-phenylalanine methyl ester [for-Met-Tic-Phe-OMe] 3 and formyl-methionyl-2-aminoxy-4-methylvaleryl-phenylalanine methyl ester [for-Met-OLeu-Phe-OMe] 4 were synthesized in order to investigate the role of the amide bond at position 2 on biological activities on human neutrophils. Only analogue 2, which keeps the NH group at position 2, was found to retain activity though sterically encumbered.

Structure activity studies on chemically modified homologues of the antibiotic phytotoxic leucinostatin A.

The synthesis and a conformational study of a number of homologues of the well known antibiotic, phytotoxic leucinostatin A are reported. The circular dichroism of all the compounds are discussed. Some conclusions on the SAR of these compounds are drawn. The influence of the alpha-helical conformation and/or the increased lipophile character on their interesting biological activities is emphasized.

Conformational studies of chemotactic HCO-Met-Leu-Phe-OMe analogues.

In order to investigate the proper peptide backbone conformation able to elicit a biological activity, HCO-Met-Pro-Phe-OMe, HCO-Met-Ψ[COO]Leu-Phe-OMe, and HCO-Met-OLeu-Phe-OMe, analogues of the prototypical chemotactic peptide HCO-Met-Leu-Phe-OMe, were studied by CD and IR techniques. The results obtained comparing biological and conformational data evidence the critical presence of (i) the NH group at position 2, (ii) a rather flexible backbone, (iii) the chemical structure of the central residue which can affect the stability of a possible active conformer.

Formyl peptide chemoattractants. Synthesis and chemotactic activity.

The tetrapeptides CHO-Met-Leu-Phe-CO-NH-(CH2)n-COOMe (n = 1-5) have been synthesized. These peptides containing an omega-amino acid residue in position 4 exhibit a very high chemotactic activity. Like the chemotactic peptide CHO-Met-Leu-Phe-OMe, these tetrapeptides in solution undoubtedly adopt an "active" conformation which allows a strong interaction with the receptor on the human polymorphonuclear leukocyte surface.

Conformational studies of synthetic tripeptide chemoattractants.

The conformational behavior of the chemotactic peptide analogs formylmethionylleucylphenylalanine methyl ester (CHO-Met-Leu-Phe-OMe) and formylmethionylleucylcyclohexylalanine methyl ester (CHO-Met-Leu-Cha-OMe) has been studied in solvents of different polarity by circular dichroism and infrared absorption. Both analogs and very probably the chemotactic peptide formylmethionylleucylphenylalanine (CHO-Met-Leu-Phe-OH) preferably adopt in solution a folded "active" conformation which allows a strong interaction with the receptor on the human polymorphonuclear leukocyte surface.

Binding of the trypsin inhibitor from white mustard (Sinapis alba L.) seeds to bovine beta-trypsin: thermodynamic study.

The effect of pH and temperature on the association equilibrium constant (Ka) for the binding of the trypsin inhibitor from white mustard (Sinapis alba L.) seeds (MTI) to bovine beta-trypsin (EC 3.4.21.4) has been investigated. On lowering the pH from 9 to 3, values of Ka for MTI binding to bovine beta-trypsin decrease thus reflecting the acid-pK and -midpoint shifts, upon inhibitor association, of two independent ionizable groups, and of a three-proton transition, respectively. At pH 8.0, values of thermodynamic parameters for MTI binding to bovine beta-trypsin are: Ka = 4.5 X 10(8)M-1, delta G0 = -11.6 kcal/mol, and delta S0 = +53 entropy units (all at 21 degrees C); and delta H0 = +4.1 kcal/mol (temperature independent between 5 degrees C and 45 degrees C). Binding properties of MTI to bovine beta-trypsin have been analyzed in parallel with those concerning macromolecular inhibitor association to serine (pro)enzymes.