E. Neuzil

The primary sequence of badger myoglobin.

Abstract The badger apomyoglobin was first submitted to tryptic digestion. The tryptic hydrolysate of whole protein was fractionated by resin chromatography and each tryptic peptide was sequenced. The apomyoglobin was also cleaved by cyanogen bromide and the resulting fragments were fractionated by gel filtration; three segments were obtained; the N-terminal segment (55 residues), the C-terminal segment (22 residues) and the median segment (76 residues). Each segment was submitted to a variety of enzymatic digestions and the complete amino acid sequence of resulting peptides was established. In addition, the whole protein and the median segment were analyzed by using a sequenator (Edman); the first 31 N-terminal residues of the intact protein and the first 28 N-terminal residues of the median segment of CNBr resulting peptide were confirmed. The complete sequence of the C-terminal segment (22 residues) was also established and confirmed by manual Edmans degradation. Between badger and harbor seal myoglobins, 19 differences out of 153 residues were found. Of these amino acid replacements, one corresponds to the exchange of two bases and 18 to the exchange of one base in the coding triplets.

Transport of inorganic phosphate inPseudomonas aeruginosa

Inorganic phosphate transport by wild-typePseudomonas aeruginosa cells grown in a phosphate-limited medium involves a biphasic process. The uptake obeys Michaelis-Menten kinetics with respective apparentKm values of 1.1 μM and 10 μM for the high- and low-affinity systems. These systems may be also differentiated by their sensitivity to osmotic shock, by their specificity towards phosphite, pyrophosphate, arsenate, and some phosphonates and also by their energy requirements. The two phosphate transport systems fromP. aeruginosa are compared with the two major systems (Pst and Pit) characterized inEscherichia coli.

Utilization of 2-aminoethylarsonic acid in Pseudomonas aeruginosa

This paper describes the metabolism, transport and growth inhibition effects of 2-aminoethylarsonic acid (AEA) and 3-aminopropylarsonic acid (APrA). The former compound supported growth of Pseudomonas aeruginosa, as sole nitrogen source. The two arsonates inhibited the growth of this bacterium when 2-aminoethylphosphonic acid (AEP) but not alanine or NH4Cl, was supplied as the only other nitrogen source. The analogy between AEA and the natural compound AEP led us to examine the in vitro and in vivo interaction of AEA with the enzymes of AEP metabolism. The uptake system for AEP (Km 6 microM) was found to be competitively inhibited by AEA and APrA (Ki 18 microM for each). AEP-aminotransferase was found to act on AEA with a Km of 4 mM (3.85 mM for AEP). Alanine and 2-arsonoacetaldehyde was generated concomitantly, in a stoichiometric reaction. In vivo, AEA was catabolized by the AEP-aminotransferase since it was able to first induce this enzyme, then to be an efficient substrate. The lower growth observed may have been due to the slowness with which the permease and the aminotransferase were induced, and hence to a poor supply of alanine by transamination.

Synthesis of 4-phosphono- and of 4-(phosphonomethyl)-dl-phenylalanine, two analogues of O-phosphotyrosine

Abstract :4-Phosphono-DL-phenylalanine 1 was synthetized from 4-bromo-DL-phenylalanine or from 4-(bromomethyl)-bromobenzene ; 4-(phosphonomethyl)-DL-phenylalanine 14 was prepared from methyl p-toluate. The interest of these phosphonic analogues arises from their possible interference in the metabolism of O-phosphotyrosine.

Recherches sur l'évolution pré-biologique III. Les acides azulmiques noirs formés à partir du cyanure d'ammonium

Abstract The authors have investigated the formation and some physicochemical properties, and discuss the structure, of black azulmic acids, polymers obtained by heating an aqueous solution of ammonium cyanide. The interest in azulmic acids is connected with the problem of pre-biological molecular evolution; the hydrolysis of azulmic acids gives rise to several natural amino acids, urea, guanidine, glycocyamine and 5-amino-4-imidazole carboxamidine.

Haemoglobins, LX. Primary structure of the major haemoglobin of the sea lamprey Petromyzon marinus (var. Garonne, Loire)

Lampreys belong to the class of Cyclostomata; practically no evolution of these Vertebrates can be noted since Paleozoïc times; lampreys thus appear as a choice material for studying several problems in the field of biochemical evolution. Several monomeric haemoglobins can be characterized in the erythrocytes of the sea lamprey (Petromyzon marinus). The major constituent was isolated by chromatography, and submitted to tryptic digestion; soluble tryptic peptides were separated by gel filtration into 5 fractions; the peptides of each fraction were isolated either by Dowex-50 chromatography or by HPLC; the insoluble core was oxidized and submitted to HPLC fractionation. The primary structure of the whole chain and of the purified tryptic peptides was determined using automatic sequencing; alignment of the peptides was achieved by homology with the previously established covalent structure of the globin of Lampetra fluviatilis. The sequence we established confirms the crystallographic data of Hendrickson and Love. Globin/haem contacts are discussed; a tentative explanation of the absence of tetramerization can be proposed after comparison with the aminoacid residues involved in alpha 1 beta 1 and alpha 1 beta 2 contacts. Petromyzon globin differs at three locations (Thr/Ser3, Leu/Met58, Thr/Ser60) from Lampetra fluviatilis globin. The monomeric chain of another Cyclostomata Myxine glutinosa, differs more considerably (88 residues). Our results corroborate recent paleontologic data which favour the separation of lampreys from hagfishes; Cyclostomata cannot be considered as a monophylic group. Finally, there is a closer relation between lamprey globin and alpha chains than between this monomeric globin and beta chains, and furthermore apomyoglobins of higher vertebrates.

Transamination non enzymatique des acides aminés phosphoniques

Abstract The authors have performed a comparative study of the non-enzymic transamination of ten aminophosphonic acids and their carboxylic analogues with some carbonyl compounds (α-oxo acids, pyridoxal, pyridoxal phosphate). An unfavourable influence of the phosphonic group towards the yield of the transamination reaction is shown by chromatography. On the other hand, a great affinity of α-aminophosphonic acids for pyridoxal phosphate is demonstrated using spectrophotometric methods. These results will emphasize the influence of the acid function, besides other structural factors, in the transamination reaction of amino acids.

Inhibition ofd-alanyl-d-alanine ligase in different bacterial species by amino phosphonic acids

A comparative study was performed on the kinetic properties and the specificity ofd-alanyl-d-alanine ligases fromPseudomonas aeruginosa, Streptococcus faecalis, andStaphylococcus aureus, using some aminophosphonic acids and related compounds.dl-I-Aminoethylphosphonic acid was shown to be a competitive inhibitor of theP. aeruginosa andS. faecalis ligases; assuming ad-form stereospecificity, its activity was nearly equal to that ofd-cycloserine. 2-Aminoethylphosphonate was found to be a weak inhibitor of the ligases, in contrast to the carboxylic analog, β-alanine. γ-Aminobutyric acid and phosphoethanolamine also exhibited some inhibitory properties.

Primary sequence of the β-chain of badger haemoglobin

Abstract Badger (Meles meles) haemoglobin was purified by paper electrophoresis and converted into globin. Chain separation was carried out on a CM-cellulose column in the presence of 8 M urea. The β-chain was aminoethylated, purified by gel filtration and submitted to tryptic digestion. A fingerprint obtained with the enzymic digests showed 17 distinct ninhydrin-positive spots from which 20 pure peptides were isolated by further electrochromatographic separations. These peptides were sequenced using Dansyl-Edman and Ptc-Edman degradation techniques. The presence of amide residues was confirmed after aminopeptidase M hydrolysis. Taking human haemoglobin β-chain as a model, the covalent structure could be completely resolved without the help of any further overlapping technique. The following substitutions were noted (badger/human, position): Ala/Pro5, Ser/Ala13, Tyr/Phe41, Asp/Glu43, Ser/Ala70, Glu/Asp73, Lys/Ala76, Asn/His77, Lys/Thr87, Lys/Arg104 and Gln/Pro125. A comparison with other haemoglobin β-chains already sequenced shows a greater similarity with dog haemoglobin, the only example of β-chain of known structure in the order of Carnivores.

Transport de l'acide amino-2-éthylphosphonique chez Pseudomonas aeruginosa

Summary 2-Aminoethylphosphonic acid (ciliatine) can be used as a source of phosphorus or nitrogen by Pseudomonas aeruginosa. The conditions of its uptake have been investigated. The transport is inducible by ciliatine itself or by its homologue, 3-aminopropylphosphonate, but neither by other phosphonic compounds nor by carboxylic or sulfonic related derivatives. The induction was not suppressed by inorganic phosphate. The transport appears to be an active process, pH and temperature dependent; it requires energy and is dependent on new protein synthesis. The uptake follows Michaelis kinetics. The substrate specificity involved in ciliatine uptake favours the existence of two different transport systems : the first one, inducible by ciliatine, was very sensitive towards different aminophosphonic acids and was competitively inhibited by inorganic phosphate and methylphosphonate; the second transport system, inducible by 3-aminopropylphosphonate, appeared less sensitive towards α-aminophosphonic acids and was non competitively inhibited by phosphate and methylphosphonate. No interactions were observed with related aminocarboxylic acids or with taurine. Some molecular structural requirements for the binding of an effector on both permeases are discussed. The regulatory function of inorganic phosphate, the chief breakdown product of ciliatine, is also emphasized.