Specific sulfonation of tyrosine, tryptophan and hydroxy-amino acids in peptides.

1. ClSO3H in trifluoroacetic acid rapidly converts serine and threonine into O-sulfate ester derivatives while tyrosine and tryptophan are converted into arylsulfonic acids. 2. H2SO4 in trifluoroacetic acid reacts more slowly with serine, threonine and tyrosine while is not able to modify tryptophan. 3. All other amino acids are perfectly stable under the above reaction conditions. 4. Peptides containing susceptible amino acid residues are specifically converted into the corresponding sulfonated derivatives in high or quantitative yield.

Purification and substrate characterization of a human enkephalin-degrading aminopeptidase.

A 5000-fold purification of the enzyme responsible for the rapid inactivation of enkephalin in human blood has been achieved: this enzyme cleaves the N-terminal tyrosine from enkephalin and from short peptides provided their first amino acid is aromatic. The enzyme, an enkephalin-degrading aminopeptidase (alpha-aminoacyl-peptide hydrolase, EC 3.4.11.11), requires a free amino group on the substrate and has a maximum activity around pH 8. Its appearance molecular weight is in the range of 80 000-90 000 and an apparent Michaelis constant of 0.4 mM was determined.

Molecular recognition between serine proteases and new bioactive microproteins with a knotted structure.

Microproteins with proteinase inhibitory activity, 28 to 30 amino acids long, with 3 disulfide bridges have been isolated from Ecballium elaterium seeds. A peptide (EETI II) was isolated and behaved as a powerful trypsin inhibitor (Kd = 10(-11) to 10(-12) M). It was sequenced, chemically synthesized and the 3-D structure determined by 2-D 1H NMR. The information gained in the process enabled us to synthesize modified derivatives with inhibitory activity towards pancreatic elastase, chymotrypsin and human leucocyte elastase (Kd = 10(-7) to 10(-9) respectively). The most striking characteristic that appeared during the synthetic approach was the unfailing ability of the 28 amino acid peptides to refold and correctly close the 3 disulfide bridges, giving in each case an active compound. These disulfide bridges are assembled in a particular knotted structure, shared by few other bioactive peptides and called the 'knottin' structure. Molecular modeling of the peptide and a comparison with the other active molecules with similar topology allowed the synthesis of a chimaeric peptide, bearing 1 active site against a seryl-protease (trypsin), and 1 against a metallo-protease (carboxypeptidase A). The bis-headed peptide was able to inhibit both enzymes separately and concomitantly.

Enkephalin-degrading activity in arthropode hemolymphe.

Enkephalin and related peptides are rapidly inactivated in Astacus fluviatilis and Limulus polyphemus hemolymphe. At least three different enzymes, an aminopeptidase, a carboxypeptidase and a peptidyl-dipeptidase, acting concomitantly on the peptide substrates have been identified. The properties of these enzymes were characterized and they were compared to similar enzymes of the vertebrate blood. The opioid peptides appear to be extremely short lived in invertebrate hemolymphe, which presumably is a metabolic barrier to the action of active peptides stronger than vertebrate blood.

Sulfonic acid enkephalin: binding specificity to rat brain opiate receptors.

We tested sulfonic-acid enkephalin, a SO3H-Tyr derivative of the Leu-enkephalin, for its binding capacity to rat brain opiate receptors, by competition against several tritiated enkephalins. Using [3H]DADLE as the competitor, we demonstrated that sulfonic-acid enkephalin binds to rat brain opiate receptors, and using [3H]DSLET and [3H]DAGO as the competitors, we demonstrated that sulfonic-acid enkephalin binds preferentially to delta-opiate receptors. The affinity ratio of sulfonic-acid enkephalin for delta-opiate receptors versus mu-opiate receptors, is considerably higher than that of the parent compound, Leu-enkephalin, and of DADLE and is similar to the affinity ratio of DSLET for the same receptors.

beta-Carboline and diazepam effect on the degradation of enkephalin by the human blood aminopeptidase.

An enkephalin-degrading aminopeptidase (alpha-amino-acyl-peptide hydrolase, EC 3.4.11.11) has been purified from human plasma and has been shown to be the principle responsible for the transient half-life of enkephalin in blood. An inhibitory effect of beta-carbolines and of 3,4-dihydro-beta-carbolines on this enzyme 'in vitro' is reported. The best inhibitor is the 3-carboxylic acid (Ki congruent to 10(-4) M), while the ester, amide, and/or peptide derivatives are less potent. Since some beta-carboline derivatives have recently been shown to possess high affinity for benzodiazepine receptors in brain, the action of diazepam on the aminopeptidase activity was tested and a relevant inhibition of the human enzyme could be demonstrated.

Immobilization of enzymes on alumina by means of pyridoxal 5'-phosphate.

A number of proteases have been immobilized on alumina in a two-step procedure: the first step converted them into semisynthetic phosphoproteins which, in the second step, spontaneously bonded to alumina through their phosphate function. The immobilized enzymes thus obtained showed the physical properties typical of the inorganic carrier and a high activity on low molecular weight substrates.

'In vivo' amplification of biological activity of tetragastrin by amino acid hydroxamates.

Rat blood was shown to contain an aminopeptidase which rapidly hydrolyses short peptides containing an aromatic amino acid as N-terminal residue. Using tetragastrin (Trp-Met-Asp-PheNH2) as substrate, we showed that some amino acid hydroxamates inhibit rat aminopeptidase activity 'in vitro' in the following order: HTrpNHOH greater than HPheNHOH much greater than HAlaNHOH. The same hydroxamates markedly enhanced the biological activity of tetragastrin 'in vivo'. The amplification of the secretory effect, correlated with the amount of the hydroxamate used, strongly suggests that these compounds can stabilize a number of active peptides in vivo by inhibiting their proteolytic degradation.

Selective retention of organic phosphate esters and phosphonates on aluminium oxide.

Compounds containing the -PO3H2 function, such as monoesters of phosphoric acid and phosphonic acids, specifically bind to aluminium oxide in aqueous solution under experimental conditions where non-phosphorylated compounds are completely desorbed. The bound organic phosphate can be specifically displaced by aqueous solution of inorganic phosphates thus allowing their separation or detection by a technique similar to that of affinity chromatography. The consequences of this finding for phosphate compound biochemistry are discussed.

Alumina-phosphate complexes for immobilization of biomolecules.

Organic compounds containing the -PO3H2 function are strongly and specifically adsorbed by aluminum oxide in water within a large range of pH. The reversible character of the interaction allows the adsorbed organic phosphates to be displaced by inorganic phosphate buffers resulting in their purification by an affinity-like chromatographic procedure. The interaction between alumina and selected multifunctional compounds containing a phosphonate group yields a chemically activated alumina-phosphate complex onto which enzymes or other molecules can be immobilized. A number of proteases immobilized on alumina through such phosphate interactions proved to be active in the presence of organic solvents. As a consequence, enzyme-catalyzed peptide synthesis in a water-limited environment and optical resolution of amino acids in water-organic solvent emulsions can be accomplished.