ALA-ALA-ALA-ALA-ALA

The investigation of the catalytic properties of astacin, a zinc-endopeptidase from the crayfish Astacus astacus L., has gained importance, because the enzyme represents a novel, structurally distinct family of metalloproteinases which also includes a human bone morphogenetic protein (BMP1). Astacin releases nitroaniline from succinyl-alanyl-alanyl-alanyl-4-nitroanilide (Suc-Ala-Ala-Ala-pNA), a substrate originally designed for pancreatic elastase. This activity was unexpected since only few metalloproteinases cleave small nitroanilide substrates, and, moreover, the primary specificity of astacin toward protein substrates is determined by short, uncharged amino-acid sidechains in the P'1-position, i.e. the new N-terminus after cleavage. The specificity constants, kcat/Km, for the release of nitroaniline from substrates of the general structure Suc-Alan-pNA (n = 2, 3, 5) and Alan-pNA (n = 1, 2, 3) increase with the number of alanine residues. The longest peptide, Suc-Ala(-)-Ala-Ala-Ala-Ala-pNA, is the only one out of eleven substrates used in this study, which is cleaved at two positions by astacin. The first cleavage yields Suc-Ala(-)-Ala and Ala-Ala-Ala-pNA. From the resulting C-terminal fragment, Ala-Ala-Ala-pNA, a second cut releases nitroaniline. The 1200-fold higher specificity constant observed for the first as compared to the second cleavage in Suc-Ala-Ala-Ala-Ala-Ala-pNA reflects the preference of astacin for true peptide bonds and also the importance of a minimum length of the substrate.(ABSTRACT TRUNCATED AT 250 WORDS)

To immobilize lipid vesicles on a polymer support, we have used a peptidic anchor with the following sequence: Ala-Ala-Leu-Leu-Leu-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-A la-Ala-Ala-Ala-Ala-Ala-Ala-Trp-Lys-Lys-Lys-Lys-Lys-Lys. This amphiphilic peptide was previously designed in our group to interact spontaneously and strongly with vesicles without perturbing their permeability. At the end of the solid-phase peptide synthesis, the peptide was left on the polymer beads and this novel polymer-peptide system was used for vesicle immobilization. It was shown that this polymer-peptide system could immobilize as much as 200 micromol of lipids per gram of dry resin. The amount of immobilized vesicles was decreased by a reduction of the proportion of the negatively charged lipids in the vesicles, indicating the importance of electrostatic interactions in the immobilization of the vesicles. The integrity of the vesicles was mostly preserved after the immobilization. This new polymer-peptide system was used easily and successfully to immobilize a membrane-bound enzyme, gamma-glutamyl transpeptidase. The activity of the membrane-bound enzyme was studied by monitoring the release of p-nitroaniline. The activity of the enzyme was still retained, even after being re-used eight times, indicating the strong immobilization of the enzyme in its active form. The polymer-peptide support could be regenerated by washing with ethanol and reused.

By using the potentiometric titration method, we have determined the pK(a) values of the two terminal lysine groups in six alanine-based peptides differing in the length of the alanine chain: Ac-Lys-Lys-NH(2) (KK), Ac-Lys-Ala-Lys-NH(2) (KAK), Ac-Lys-Ala-Ala-Lys-NH(2) (KAK2), Ac-Lys-Ala-Ala-Ala-Lys-NH(2) (KAK3), Ac-Lys-Ala-Ala-Ala-Ala-Lys-NH(2) (KAK4), and Ac-Lys-Ala-Ala-Ala-Ala-Ala-Lys-NH(2) (KAK5) in aqueous solution. For each compound, the model of two stepwise acid-base equilibria was fitted to the potentiometric-titration data. As expected, the pK(a) values of the lysine groups increase with increasing length of the alanine spacer, which means that the influence of the electrostatic field between one charged group on the other decreases with increasing length of the alanine spacer. However, for KAK3, the pK(a1) value (8.20) is unusually small and pK(a2) (11.41) is remarkably greater than pK(a1), suggesting that the two groups are close to each other and, in turn, that a chain-reversal conformation is present for this peptide. Starting with KAK3, the differences between pK(a1) and pK(a2) decrease; however, for the longest peptide (KAK5), the values of pK(a1) and pK(a2) still differ by about 1 unit, i.e., by more than the value of log(10) (4) = 0.60 that is a limiting value for the pK(a) difference of dicarboxylic acids with increasing methylene-spacer length. Consequently, some interactions between the two charged groups are present and, in turn, a bent shape occurs even for the longest of the peptides studied.