Insoluble biodegradative potential of the venice lagoon.

The sediment phase of the Venice lagoon within an area of shallow water, the Palude della Rosa, was studied and three insoluble enzymatic activities (cellulase, phosphatase and urease) were found to be linked to the inorganic phase. These immobilized enzymes were more resistant to environmental changes, even extreme ones, compared to their soluble counterparts. The evolution of their activity with pH, temperature and seasonal variation was investigated. While pH related activities showed the usual behaviour, the resistance to temperature was extremely high and seasonal variation was dependent on immobilization. These enzymatic activities could be used as a diagnostic factor for the ecosystem, since their presence is related to the nature of the waste products.

Insect immunity: two proteinase inhibitors from hemolymph of Locusta migratoria.

Two protease inhibitors were isolated from the plasma of Locusta migratoria and sequenced. They were 35 and 36 amino acids long and revealed very little similitude for the protease inhibitors isolated from other arthropods. They inhibit the proPhenoloxidase Phenoloxidase proteolytic activation cascade in hemocyte extracts of the same insect. This inhibiting activity resulted in a lower production of PO, a key enzyme for the defence mechanism in arthropods. Both peptides however showed a strong in vitro inhibiting activity toward alpha-chymotrypsin and elastase, LMCI I inhibits the human leukocyte enzyme while LMCI II mostly the pancreatic one, a difference explainable on the basis of the active site sequence changes.

Purification of a protease inhibitor which controls prophenoloxidase activation in hemolymph of Locusta migratoria (insecta).

A protein which inhibits the prophenoloxidase----phenoloxidase (EC 1.14.18.1) proteolytic activation in hemocyte extracts of Locusta migratoria was isolated from the plasma of the same insect and partially characterized. It shows a molecular weight of 14,000, an inhibiting activity toward the cascade system in the insect hemocytes, which resulted in a lower production of phenoloxidase, a key enzyme for the defence mechanism in arthropods. To identify the specificity of the Locusta inhibitor and consequently the specificity of its target enzyme, inhibitory tests were performed against a number of known serine-proteases. A strong in vitro inhibiting activity toward chymotrypsin and, to a lesser extent, toward human leukocyte elastase was present, while trypsin, Carlsberg subtilisin, human thrombin and pancreatic elastase failed to react. The lack of trypsin inhibition by the isolated inhibitor suggested that the trypsin-catalysed activation of the system in the hemocyte extract takes place under different controls or at an earlier stage of the cascade. The N-terminal sequence of the inhibitor reveals that this molecule is different from the protease inhibitors isolated from other arthropods.

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.

N-terminal domain of pepsin as a model for retroviral dimeric aspartyl protease.

Autolysis of porcine pepsin at pH 4 affords a derivative possessing intrinsic proteolytic activity. This derivative was isolated by alumina pseudo-affinity chromatography and gel-filtration and was found to result from the tight association of two identical molecules, 135 amino acids long, emerging from the N-terminal domain of pepsin. This finding emphasizes a similarity with the only aspartyl-proteases known to act as dimers, the retroviral proteases.

Design and chemical synthesis of a 32 residues chimeric microprotein inhibiting both trypsin and carboxypeptidase A.

A chimeric peptide, 32 amino acids long, bearing two active sites, one inhibiting trypsin (Kd = 1.8 10(-9) M), one carboxypeptidase A (Kd = 3 10(-9) M), was designed and synthesized. It is a "squash inhibitor" (EETI II, 28 amino acids) elongated with the 4 amino acids from the C-terminus of the potato carboxypeptidase inhibitor. It has 3 disulfide bridges assembled in the particular knotted topology shared by the two inhibitors, by conotoxin omega, and possibly by E. coli enterotoxin ST1b.

Active site chemical mutagenesis of Ecballium elaterium trypsin inhibitor II: new microproteins inhibiting elastase and chymotrypsin.

Seven microproteins analogous to Ecballium elaterium Trypsin Inhibitor II. were synthesized. The study of their inhibiting power showed a change in selectivity from trypsin to elastase for 5 of the compounds and to alpha-chymotrypsin for another one. A striking characteristic that appeared during this synthetic approach was the ability of the 28 amino acid peptides to refold and close correctly the 3 disulfide bridges, giving in each case an active compound.

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.

Protease inhibitors from Ecballium elaterium seeds.

Several protease inhibitors were found in the seeds of a Cucurbitacea, Ecballium elaterium, and were separated from one another by affinity and molecular sieve chromatography. Three main trypsin isoinhibitors were purified by ion-exchange chromatography and the sequence of the major one, EETI II, was elucidated and compared with other inhibitors of the squash family. It is a peptide of M.W. 3020 of strong inhibitory activity (Ka = 8 x 10(11) M-1) against trypsin, showing high Gly content, six half-cystine residues, but devoid of histidine, threonine, tryptophan, and tyrosine residues.

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.

Peptide and ester synthesis in organic solvents catalyzed by seryl proteases linked to alumina.

Trypsin and alpha-chymotrypsin were immobilized to alumina-phosphocolamine complex, activated by glutaraldehyde. The immobilized enzymes show a great stability toward organic solvents miscible or immiscible with water. In the presence of a low concentration of water, the immobilized enzymes catalyzed transesterification reactions as well as peptide synthesis. The synthesized peptides were stable toward the immobilized enzymes.

Chymotrypsin catalysed synthesis of fluorescent amino acid amide and peptide derivatives.

Chymotrypsin catalyses a condensation reaction between 1-methyl-3,4-dihydro-beta-carboline-3-methyl carboxylate and amino acid amides or peptides, yielding fluorescent derivatives. During the peptide bond formation, the enzyme ensures the reaction's steric control of both carboxyl and amino components.

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.

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.

'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.

An N-protected gamma-aminobutyric acid dipeptide with anticonvulsant action.

N-Pivaloyl-leucyl-gamma-aminobutyric acid (PLG) is a synthetic dipeptide with a partition coefficient of 1.67 in an ethyl acetate/water system that partially inhibits the synaptosomal uptake and activates the release of [U-14C]gamma-aminobutyric acid [( U-14C]GABA). The displacement of GABA from crude synaptic membranes by PLG occurs with an IC50 of 10(-5) M. The compound has the capacity to cross the blood-brain barrier and increase central GABA levels. Its ED50 on cardiazol -induced convulsions is 60-65 mg/kg. PLG is resistant to hydrolysis by chymotrypsin and partially inhibits the proteolytic activity of trypsin.

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.

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.