The crystal structure of citrate synthase from the thermophilic Archaeon Sulfolobus solfataricus (optimum growth temperature ¼ 85°C) has been determined, extending the number of crystal structures of citrate synthase from different organisms to a total of five that span the temperature range over which life exists (from psychrophile to hyperthermophile). Detailed structural analysis has revealed possible molecular mechanisms that determine the different stabilities of the five proteins. The key to these mechanisms is the precise structural location of the additional interactions.As one ascends the temperature ladder, the subunit interface of this dimeric enzyme and loop regions are reinforced by complex electrostatic interactions, and there is a reduced exposure of hydrophobic surface. These observations reveal a progressive pattern of stabilization through multiple additional interactions at solvent exposed, loop and interfacial regions.
A new bacterial method for determining amino acids in protein foods is described. Instead of the ‘natural’microbial auxotrophs e.g. Tetrahymena, Streptococcus, and Leuconostoc, currently used for such assays, an ‘artificial’mutant is used, viz. an auxotroph of Escherichia coli. Test proteins (Bovine serum albumin, legume and maize meals) were predigested with a mixture of pronase and intestinal peptidases, the efficiency and extent of proteolysis being monitored by pH stat titration. Final digests were examined by Sephadex gel filtration to ensure that all protein cleavage products were small enough to pass through the E. coli cell wall and to reach its cyto‐plasmic amino acid and peptide permeases. The lysine content of the meals, as determined from the growth of an E. coli lysine auxotroph upon the digests, was found to be greater than 90° of the lysine determined chemically in acid hydrolysates. Practical and theoretical advantages of using this latter type of bacterium rather than the fastidious species are discussed. In addition, the particular value of using an intestinal bacterium like E. coli to assay nutritional availability of amino acids is considered in relation to its normal utilization of digested protein foods in vivo, and the similarities between its amino acid and peptide permeases and those of the intestine.
Temperature-programmed calcination of basic chromium(III)
acetate impregnated onto a high surface area
silica support and chromium(III) acetylacetonate dry-blended with
silica has been studied by quadrupole
mass spectrometry and infrared spectroscopy. In both cases the
same reactive intermediate has been identified.
A direct study of peptide uptake by Escherichia coli was made using a fluorescent procedure. After incubation with the bacteria, peptides remaining in the medium were dansylated, separated chromatographically, and quantitated from their fluorescent intensities and/or from their incorporated radioactivity when tritiated dansyl derivatives were prepared. Peptide uptake was apparently not regulated and proceeded continuously until complete, with the absorbed peptides undergoing rapid intracellular hydrolysis and the excess amino acid residues leaving the cell. Thus, peptide uptake and amino acid exodus occur concurrently. However, peptidase-resistant substrates, e.g. triornithine and glycylsarcosine, which can be similarly estimated in cell extracts, were accumulated about 1,000-fold. The influence of amino acid composition and chain length on rates of transport was assessed. Different strains of E. coli showed variability in their rates of di-and oligopeptide transport. With respect to energy coupling, both the diand oligopeptide permeases behaved like shock-sensitive transport systems.
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