A synthetic calcium-chelating L-glutamyl-L-serine phosphate (Glu-Ser.P) enhances calcium absorption in the rat.

A new synthetic procedure was used to prepare L-glutamyl-L-serine phosphate (Glu-Ser.P) with a good yield. The phosphopeptide is able to chelate calcium ion as shown by conductimetric titration and through the use of a calcium-specific electrode. Intestinal absorption of radioactive calcium is enhanced in rats fed 45Ca and Glu-Ser.P.

One-step conversion of amino acids into N-menthyloxycarbonyl alkyl ester derivatives for chiral gas chromatography.

Amino acids dissolved in aqueous methanol (or ethanol) and treated with optically active menthyl chloroformate were converted into N-menthyloxycarbonyl methyl (or ethyl) ester derivatives within a few minutes at room temperature. The obtained diastereomeric derivatives, with the exception of arginine and histidine, had suitable gas chromatographic properties allowing enantiomeric analysis of a large number of proteinogenic and synthetic amino acids.

Racemization of alpha-amino acid esters by aliphatic ketones in the presence of carboxylic acids.

Amino acid esters are racemized by dissolution in a mixture of aliphatic ketones and carboxylic acids. The racemization rate mainly depends on the structure of the amino acid and on the kind of ketone and carboxylic acid used, the best racemizing medium being acetone containing 15% acetic acid. The mechanism of the racemization and the practical consequences of this study in the optical resolution field 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.

Synthesis and some properties of a homologous series of beta-carboline-3-carboxylic esters.

A homologous series of esters of beta-carboline-3-carboxylic acid was prepared by a new synthetic procedure. The first members of the series are convulsant or pro-convulsant agents while those with acyl residues longer than C4 show anticonvulsant activity. The affinity of the members of the series for the benzodiazepine receptor from rat brain decreases with the increasing chain length of the esters. The pentyl ester was studied in particular for its anti-convulsant effect on rats and mice treated with cardiazol. The compound also acts as a membrane stabilizer by inhibiting red cell hypotonic hemolysis and rat brain Na/K ATPase; its LD50 value is 12.5 mg/Kg and of particular interest is its lack of inhibitory effect on memory retention in mice at doses at which diazepam has inhibitory effects.

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.

Dopamine-depleting activity of L-3,4-(dioxyphenylacetyl)-phenylalanine.

Dioxyphenylacetyl-L-DOPA was synthesized and shown to be resistant toward oxidative cyclization in the presence of plasma and brain extracts. This new DOPA derivative is slightly inhibitory towards monoamine oxidase (IC50 2.5 mM) and the synaptosomal uptake of dopamine (IC50 0.35 mM) and acts as an antagonist of haloperidol (IC50 0.6 microM) in displacing 3H-spiroperidol from isolated dopamine receptors. The substance, although more hydrophobic than DOPA, overcomes the blood-brain barrier to a lower extent than DOPA and lowers the levels of dopamine and norepinephrine in the left and right hemisphere of mice treated with it for 10 days at a dose of 10 mg/kg/day. Dioxyphenylacetyl-L-DOPA inhibits red cell hypotonic hemolysis and brain Na/K+-ATPase activity in vitro and potentiates the barbiturate-induced sleep.

Effect of 2-pyrrolidone on the concentration of GABA in rat tissues.

The effects of 2-pyrrolidone, a cyclic lactam of GABA, were studied on blood and organ levels of 2-pyrrolidone, GABA, glutamic acid, glutamate decarboxylase (GAD) and GABA-transaminase (GABA-T). When administered i.p., the only significant effects observed were increases of brain and liver 2-pyrrolidone. In contrast, regular oral administration for 7 months produced significant increases of GABA and glutamic acid in brain and of glutamic acid alone in liver while GAD decreased in brain and increased in liver; GABA-T was unchanged. A new method for the synthesis of radioactive 2-pyrrolidone was set up and the enzymatic conversion of 2-pyrrolidone to GABA was measured by an original procedure. The results obtained in vitro by this method on the conversion of 2-pyrrolidone to GABA catalyzed by tissue slices, together with the observed inhibition of the GABA-dependent oxygen consumption by 2-pyrrolidone, partially explain the effects of the oral administration.

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.

[Simple compounds with high pharmacologic potential: beta-carbolines. Origins, syntheses, biological properties]. / Des structures simples aux potentialités pharmacologiques élevées: les beta-carbolines. Origines, synthèses, propriétés biologiques.

We reviewed the origins, the synthetic pathways and the biological properties of beta-carbolines, the condensation products of tryptophan and indole alkylamines with aldehydes. They were found in many plants, some of which have been used as hallucinogens. They also occur as minor constituents in tobacco smoke. In mammalian body, beta-carboline derivatives occur normally in plasma, platelets and urine, moreover it seems that some are formed in human body after alcohol intake. Due to interesting biological effects described in recent years (inhibition of monoamine oxidase, binding to benzodiazepine receptors, comutagenic and carcinogenic properties, 5-hydroxy tryptamine uptake inhibition), many attempts were made to prepare beta-carbolines starting from various indole derivatives. We reviewed the published methods up to 1975 and summarized the main patents related with pharmacological properties of synthetic beta-carbolines.

'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.

Enkephalin-degrading activity in Xenopus laevis tadpoles and adults blood.

The enkephalin-degrading enzyme (alpha-aminoacyl-peptide hydrolase,EC 3.4.11.11) responsible for the rapid inactivation of enkephalins in human blood was purified and compared to the enkephalin-degrading system of Xenopus laevis tadpoles and adult blood. The specificity and the kinetic constants of the Xenopus enzyme(s) were determined after partial purification. Even if remarkable similarities between the Xenopus and the human enzyme exist, still they show differences in specificity towards peptides whose N-terminal is Phe. Amphibians are able to manufacture enkephalins and the present work shows that they are endowed with an enkephalin-degrading system comparable to the soluble one of human blood.

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.

Effect of Nin-formylation of the tryptophan residue on gastrin (HG-13) binding and on gastric acid secretion.

The biologically active iodinated [Leu11]human gastrin-13 was found to exhibit high affinity for gastric mucosal cell receptors. We compared its biological activity on rat stomach and its receptor affinity on rat gastric mucosal cells isolated with pronase with those of a reversibly modified derivative obtained by direct Nin-formulation of the Trp10 residue. The results strongly suggested the involvement of the indole-NH group in both the specific binding of gastrin and the triggering of acid secretion.

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.