Arginine degradation in Pseudomonas aeruginosa mutants blocked in two arginine catabolic pathways

Summary Pseudomonas aeruginosa mutants defective in agmatine utilization (agu) were isolated. The genes encoding agmatine deiminase (aguA) and N-carbamoylputrescine amidinohydrolase (aguB) were 98% cotransducible and mapped between gpu and ser-3 in the 30 min region of the chromosome. Constructed agu arc double mutants (blocked in the arginine decarboxylase and arginine deiminase pathways) used arginine efficiently as the sole carbon and nitrogen source. This suggests the existence of a further arginine catabolic pathway in P. aeruginosa. The mapping data of this study confirm that in P. aeruginosa the chromosomal genes with catabolic functions do not show supraoperonic clustering as found in P. putida.     

Metabolomics of reef benthic interactions reveals a bioactive lipid involved in coral defence

Holobionts are assemblages of microbial symbionts and their macrobial host. As extant representatives of some of the oldest macro-organisms, corals and algae are important for understanding how holobionts develop and interact with one another. Using untargeted metabolomics, we show that non-self interactions altered the coral metabolome more than self-interactions (i.e. different or same genus, respectively). Platelet activating factor (PAF) and Lyso-PAF, central inflammatory modulators in mammals, were major lipid components of the coral holobionts. When corals were damaged during competitive interactions with algae, PAF increased along with expression of the gene encoding Lyso-PAF acetyltransferase; the protein responsible for converting Lyso-PAF to PAF. This shows that self and non-self recognition among some of the oldest extant holobionts involve bioactive lipids identical to those in highly derived taxa like humans. This further strengthens the hypothesis that major players of the immune response evolved during the pre-Cambrian.     

The immunochemical detection and quantitation of intracellular ubiquitin-protein conjugates

ATP, ubiquitin-dependent proteolysis proceeds through covalent intermediates between target proteins destined for degradation and the 8,600-Da polypeptide ubiquitin. The ubiquitin moiety therefore represents a sensitive immunological marker for the specificity and function of this novel post-translational modification. Methods are described for the immunochemical detection of ubiquitin conjugates immobilized on nitrocellulose filters following electrophoretic transfer from sodium dodecyl sulfate-polyacrylamide gels. A further modification allows quantitation of conjugated ubiquitin to the exclusion of free polypeptide. Comparisons of conjugate pools in rabbit reticulocytes and erythrocytes demonstrate that 83 +/- 3% and 31 +/- 0.2%, respectively, of total intracellular ubiquitin exists covalently bound to target proteins. Similar large proportions of conjugated ubiquitin were found in three tissue culture cell lines. Subcellular fractionation revealed that 25% of total ubiquitin conjugates of reticulocytes sediment with the 22,000 X g stromal fraction with the remainder found in the 100,000 X g supernatant. In contrast, significant levels of erythrocyte ubiquitin conjugates occur only in the 100,000 X g supernatant, suggesting ubiquitin-mediated proteolysis actively degrades stromal components lost during terminal maturation. Reticulocytes retain their full complement of active ubiquitin during maturation indicating the concomitant decline in energy-dependent proteolysis does not result from ubiquitin inactivation. That the lower level of ubiquitin conjugates and the accompanying rate of energy-dependent proteolysis in erythrocytes is a consequence of limited substrate availability is suggested by observed increases in conjugate pools and induction of specific ubiquitin-protein adducts on incubation with either phenylhydrazine or sodium nitrite.     

Studies on the role of methionine in porcine pancreatic phospholipase A2.

The unique methionine-15 residue located at the N-terminal site of iso- or beta-phospholipase A2 from porcine pancrease has been specifically carboxymethylated with iodoacetic acid. The modification results in a complete inactivation of the enzymatic activity toward micellar and monomeric substrates. Spectroscopic measurements reveled that the carboxymethylated protein still binds Ca2+ and monomeric substrates with comparable affinities as the native enzyeme. The active site histidine-54 residue in the modified enzyme shows a reactivity toward the active site-directed irreversible inhibitor p-bromophenacylbromide which is identical to that of the native enzyme. The alkylated protein, however, has lost its ability to bind to lipid-water interfaces. Although circular dichroic spectra of the carboxymethylated enzyme display some changes in the tertiary structure as compared with the native enzyme, the alpha-helix content remains rather constant. It is concluded that carboxymethylation of methionine-15 destroys the interface recognition site but has only limited influence on the active site of the molecule. Therefore, it seems that methionine-15 is not involved in the catalytic events but that this residue is part of the interface recognition site which embraces the N-terminal hydrophobic part of the enzyme: Ala-Leu-Trp-Gln-Phe-Arg-Ser-Met.     

Competitive inhibition of lipolytic enzymes. II. Preparation of 'monoacylamino' phospholipids

This paper describes the synthesis of a number of phosphatidylcholines and phosphatidylglycols, in which one fatty acyl ester group is replaced by an acylamino function. The phospholipids, both of the alpha- and beta-type, are prepared in racemic and enantiomeric pure forms.