5-A Fourier map of gramicidin A phased by deuterium-hydrogen solvent difference neutron diffraction

Crystals of ion-free gramicidin A (P212121: a = 24.61, b = 32.28, c = 32.52) have been investigated using neutron diffraction. A difference analysis of crystals soaked in ethanol/H2O as opposed to ethanol-d6/D2O has led to single isomorphous replacement Fourier projections of the structure at 5-A resolution. The gramicidin dimer appears to be a 32-A-long cylinder oriented parallel to the c-axis in these crystals.     

Kinetics of the activation of rat liver pyruvate kinase by fructose 1,6-disphosphate and methods for characterizing hysteretic transitions

1. Kinetics of fructose 1,6-diphosphate activation of liver pyruvate kinase type I inhibited with MgATP and l-alanine are described as a function of enzyme and fructose 1,6-diphosphate concentrations. These results can be explained by a single pseudo-first-order transition of the enzyme into an active form, independent of the enzyme concentration. This rate constant, k(app.)=0.24s(-1) with 0.02mm-fructose 1,6-diphosphate (t(0.9) approximately 10s where t(0.9) is the time for 90% conversion), is an increasing function of fructose 1,6-diphosphate concentration far beyond that needed to maximally activate enzyme equilibrated with fructose 1,6-diphosphate (about 20mum). 2. The model equations are best analysed with numerical techniques which are described. These techniques are useful in studying similar slow transients frequently observed in stopped-flow studies of enzymes. 3. Shorter transients (t(0.9)=0.5-1.5s) were observed in the kinetic response of the enzyme to the addition of MgATP or phosphoenolpyruvate, but were not further characterized.     

The `neurotoxicity'' of l-2,4-diaminobutyric acid

The neurolathyrogen l-2,4-diaminobutyric acid is concentrated by liver, and liver damage can yield neurotoxicity; thus the neurotoxicity caused by this compound may be due to liver damage followed by secondary brain damage. 1. The intraperitoneal administration of toxic doses of l-2,4-diaminobutyric acid to rats resulted in hyperirritability, tremors and convulsions in 12-20hr. and increased the concentration of ammonia of blood and brain slightly and the concentration of glutamine of brain two- to three-fold. By contrast, toxic doses of l-homoarginine, l-lysine, l-leucine and ammonium acetate caused dyspnoea, extreme prostration, and in some cases coma in 15-30min., and increased the concentration of ammonia of blood significantly and the concentration of glutamine of brain slightly. These results indicate that l-2,4-diaminobutyric acid caused a chronic ammonia toxicity, whereas the other amino acids and ammonium acetate resulted in an acute ammonia toxicity. 2. Liver slices from l-2,4-diaminobutyric acid-treated animals and normal liver slices preincubated with l-2,4-diaminobutyric acid utilized ammonia and formed urea at a lower rate than control slices from normal rats. 3. l-2,4-Diaminobutyric acid inhibited competitively ornithine carbamoyltransferase of rat liver homogenates, thus demonstrating that this reaction is a primary site of toxicity for this neurolathyrogen. Although we were unable to show marked elevations of blood ammonia concentration after treatment with l-2,4-diaminobutyric acid, these results are interpreted to mean that ammonia utilization (urea synthesis) in liver is inhibited by l-2,4-diaminobutyric acid and that at least part of the neurotoxicity is due to a prolonged slight increase in body ammonia concentration.     

Utilization in vivo of glucose and volatile fatty acids by sheep brain for the synthesis of acidic amino acids

1. Free glutamic acid, aspartic acid, glutamic acid from glutamine and, in some instances, the glutamic acid from glutathione and the aspartic acid from N-acetyl-aspartic acid were isolated from the brains of sheep and assayed for radioactivity after intravenous injection of [2-¹⁴C]glucose, [1-¹⁴C]acetate, [1-¹⁴C]butyrate or [2-¹⁴C]propionate. These brain components were also isolated and analysed from rats that had been given [2-¹⁴C]propionate. The results indicate that, as in rat brain, glucose is by far the best precursor of the free amino acids of sheep brain. 2. Degradation of the glutamate of brain yielded labelling patterns consistent with the proposal that the major route of pyruvate metabolism in brain is via acetyl-CoA, and that the short-chain fatty acids enter the brain without prior metabolism by other tissue and are metabolized in brain via the tricarboxylic acid cycle. 3. When labelled glucose was used as a precursor, glutamate always had a higher specific activity than glutamine; when labelled fatty acids were used, the reverse was true. These findings add support and complexity to the concept of the metabolic `compartmentation' of the free amino acids of brain. 4. The results from experiments with labelled propionate strongly suggest that brain metabolizes propionate via succinate and that this metabolic route may be a limited but important source of dicarboxylic acids in the brain.     

Energetics of gramicidin hybrid channel formation as a test for structural equivalence. Side-chain substitutions in the native sequence.

To determine whether amino acid side-chain substitutions in linear gramicidins after the structure of membrane-spanning channels formed by the modified peptides, we have developed a quantitative measure of structural equivalence of the peptide backbone among gramicidin channels based on functional (single-channel) measurements. The experiments exploit the fact that gramicidin channels are symmetrical dimers, and that channels formed by different gramicidin analogues can be distinguished on the basis of their single-channel current amplitudes or durations. It is thereby possible to determine whether hybrid channels can form between chemically dissimilar peptides, i.e. whether the peptides can adapt to each other. Further, since the relative rates of channel formation as well as the relative concentrations of pure and hybrid channel types can be measured in the same membrane, these experiments provide a quantitative measure of the energetic cost of hybrid channel formation relative to the formation of the pure channels. For a wide variety of different side-chains, we find that substitutions as extreme as glycine to phenylalanine at position 1, at the join between the two monomers in a membrane-spanning dimer, incur no energetic cost for channel formation, which implies that channels formed by each of the modified peptides are structurally equivalent. In addition, the average durations of the hybrid channels (except those having tyrosine or hexafluorovaline at position 1) are intermediate to the average durations of the respective pure channel types, thus providing further evidence for structural equivalence among channels formed by sequence-substituted gramicidins.     

Colonization factors of diarrheagenicE. coli and their intestinal receptors

WhileEscherichia coli is common as a commensal organism in the distal ileum and colon, the presence of colonization factors (CF) on pathogenic strains ofE. coli facilitates attachment of the organism to intestinal receptor molecules in a species- and tissue-specific fashion. After the initial adherence, colonization occurs, and the involvement of additional virulence determinants leads to illness. EnterotoxigenicE. coli (ETEC) is the most extensively studied of the five categories ofE. coli that cause diarrheal disease, and has the greatest impact on health worldwide. ETEC can be isolated from domestic animals and humans. The biochemistry, genetics, epidemiology, antigenic characteristics, and cell and receptor binding properties of ETEC have been extensively described. Another major category, enteropathogenicE. coli (EPEC), has virulence mechanisms, primarily effacement and cytoskeletal rearrangement of intestinal brush borders, that are distinct from ETEC. An EPEC CF receptor has been purified and characterized as a sialidated transmembrane glycoprotein complex directly attached to actin, thereby associating CF-binding with host-cell response. Three, additional categories ofE. coli diarrheal disease, their colonization factors and their host cell receptors are discussed. It appears that biofilms exist in the intestine in a manner similar to oral bacterial biofilms, and thatE. coli is part of these biofilms as both commensals and pathogens.Abbreviations CF colonization factor - CFA Colonization Factor Antigen - CS coli-surface-associated antigen - EAggEC enteroaggregativeE. coli - ECDD E. coli diarrheal disease - EHEC enterohemorrhagicE. coli - EIEC enteroinvasiveE. coli - EPEC enteropathogenicE. coli - ETEC enterotoxigenicE. coli - Gal galactose - GalNAc N-acetyl galactosamine - LT heat-labile toxin - NeuAc N-acetyl neuraminic acid - PCF Putative colonization factor - RBC red blood cells - SLT Shiga-like toxin - ST heat-stable toxin     

Helical channels in crystals of gramicidin A and of a cesium--gramicidin A complex: an x-ray diffraction study.

X-ray crystallographic studies of gramicidin A crystallized from methanol (P2₁) and ethanol (P2₁2₁2₁), and of a Cs⁺ gramicidin A complex crystallized from methanol (P222₁, P2₁2₁2 or P2₁2₁2₁) are reported here. The asymmetric unit consists of two molecules of gramicidin A in the native crystals and four molecules in the cesium complex crystal. Patterson analyses show that gramicidin A in these crystals forms a cylindrical helical channel. In the two types of native gramicidin crystals, the diameter of this channel is about 5 å and its length is about 32 å. Cesium ions are bound inside this channel in crystals of the cesium-gramicidin A complex. The channel in this complex is considerably shorter (26 Å) and wider (6·8Å) than in the native forms. The Patterson maps of these three crystal forms are compatible with either the single-stranded β-helix (Urry, 1971) or the double-stranded parallel or anti-parallel, β-helix (Veatch et al., 1974).     

Photoaffinity labelling of juvenile hormone binding proteins in the cockroach Leucophaeamaderae

The synthesis and testing of several diazocarbonyl JH analogs (diazo JHA) which act as photoaffinity labels for insect juvenile hormone binding proteins are described. The best competitor, 10,11-epoxyfarnesyl diazoacetate, has been shown to irreversibly reduce [³H]-JH III binding to both ovarian and hemolymph JHBP from Leucophaeamaderae after irradiation at 254 nm for 20 seconds. No loss of activity was observed after incubation of JHBP and diazo JHA without irradiation. Protection from photoinactivation by diazo JHA II was achieved by the presence of an equimolar amount of JH III during the photolysis. Photoaffinity labeled proteins show loss of binding capacity without alteration of the binding affinity. This is the first example of the use of a photoaffinity label in the study of JH action on a molecular level, and may become a valuable tool in the elucidation of JH-receptor-chromatin interactions.     

Ovarian maturation in Leucophaea maderae: Juvenile hormone regulation of thymidine uptake into follicle cell DNA

Our research demonstrates that juvenile hormone (JH I) stimulates thymidine incorporation into ovarian follicle cell DNA in the ovoviviparous cockroach, Leucophaea maderae.A rapid, quantitative method for monitoring ³H-thymidine incorporation into ovarian DNA, in vitro, is described. Cultured ovarian tissue from L. maderae incorporates ³H-thymidine into DNA at a linear rate between 16 and 120 min; analysis of the incorporated label revealed at least 98% of it to be in DNA.Using L. maderae females that had been mated 7 days after adult emergence, we monitored the following biochemical phenomena during the 18–22 day period of terminal oöcyte growth: (1) ³H-thymidine incorporation into ovarian DNA: (2) general protein synthesis in fat body; and (3) specific fat body vitellogenin synthesis.Decapitation of mated females with maturing oöcytes arrested both ovarian DNA synthesis and fat body vitellogenin synthesis. Substantial restoration of both types of synthesis was induced by injection of JH I. The resumption of thymidine incorporation into DNA was localized in the follicular epithelium of the terminal oöcyte.In decapitated virgin females, injection of JH I stimulated oöcyte growth and ³H-thymidine incorporation into ovarian DNA. Dose and time response curves indicate that peak stimulation of ovarian DNA synthesis occurred between 72 and 96 hr after administration of a single optimal dose of 25 μg JH I. The concurrent manifestation of ³H-thymidine uptake into ovarian DNA and activity within the fat body indicates that a similar hormonal mode of action may be operative with respect to both tissue types in virgin females.