Dipole moments of random coil polypeptides

The Rotational Isomeric States model is applied to calculate dipole moments of polypeptides of the twenty natural α-amino acids in the random coil state. Dipole moments of each repeat unit (μ_i), are evaluated using a quantum mechanics procedure. Dipole moment ratios ($\text{D}{}_{\mathrm{x}}\text{=}\text{〈μ}{}^2\text{〉}\text{x}\text{μ}{}_{\mathrm{i}}{}^2\text{,}\text{x = number of repeat units}$) of homopolypeptides are calculated and extrapolated to x →?. With a few exceptions, D_? = 0.36 ± 0.1. Ten actual proteins and three enzymes are also studied; their dipole ratios (D_x′ =〈μ〉/x) range from 7.34 to 10.57 in 10^?59 C² m² (6.6–9.5 D²). Diffferences in the values of D_x′ are due mainly to the different contributions, μ_i, of the amino acid residues contained in each polymer, whereas the sequence of amino acids has a very minor effect.     

ADH and phylogenetic relationships ofDrosophila lebanonensis (Scaptodrosophila)

Summary Increasing data onDrosophila alcohol dehydrogenase (ADH) sequences have made it possible to calculate the rate of amino acid replacement per year, which is 1.7×10^–9. This value makes this protein suitable for reconstructing phylogenetic relationships within the genus for those species for which no molecular data are available such asScaptodrosophila. The amino acid sequence ofDrosophila lebanonensis is compared to all of the already knownDrosophila ADHs, stressing the unique characteristic features of this protein such as the conservation of an initiating methionine at the N-terminus, the unique replacement of a glycine by an alanine at a very conserved position in the NAD domain of all dehydrogenases, the lack of a slowmigrating peptide, and the total conservation of the maximally hydrophilic peptide. The functional significance of these features is discussed.Although the percent amino acid identity of the ADH molecule inDrosophila decreases as the number of sequences compared increases, the conservation of residue type in terms of size and hydrophobocity for the ADH molecule is shown to be very high throughout the genusDrosophila. The distance matrix and parsimony methods used to establish the phylogenetic relationships ofD. lebanonensis show that the three subgenera,Scaptodrosophila, Drosophila, andSophophora separated at approximately the same time.     

Peptide Subunits of γ-Aminobutyric AcidA/Benzodiazepine Receptors from Bovine Cerebral Cortex

The gamma-aminobutyric acidA/benzodiazepine receptor complexes from bovine cerebral cortex were purified by immunoaffinity chromatography, and the main component peptide subunits were characterized. The peptide band originally thought to be a single beta subunit [57,000 Mr band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)] is composed of at least four different peptides of 54,000-57,000 Mr. Two peptides of 55,000 and 57,000 Mr were recognized by the beta subunit-specific monoclonal antibody 62-3G1. Peptides in the range of 54,000-57,000 Mr were photoaffinity-labeled with [3H]muscimol. A different 57,000 Mr peptide was photoaffinity-labeled by [3H]flunitrazepam, but neither was recognized by the monoclonal antibody 62-3G1 nor photoaffinity-labeled with [3H]muscimol. Some peptides could be identified by their differential mobility shift in SDS-PAGE after treatment with endoglycosidase H. Two additional subunit peptides of 51,000 and 53,000 Mr were also photoaffinity-labeled by [3H]flunitrazepam and reacted with antiserum A. However, the 57,000 Mr peptide that also was photoaffinity-labeled by [3H]flunitrazepam did not react with antiserum A.     

Modulation of Quinolinic and Kynurenic Acid Content in the Rat Brain: Effects of Endotoxins and Nicotinylalanine

Quinolinic acid, an endogenous excitotoxin, and kynurenic acid, an antagonist of excitatory amino acid receptors, are believed to be synthesized from tryptophan after the opening of the indole ring. They were measured in the rat brain and other organs using gas chromatography-mass spectrometry or HPLC. The enzyme indoleamine 2,3-dioxygenase, capable of cleaving the indole ring of tryptophan, was induced by administering bacterial endotoxins to rats, which significantly increased the brain content of both quinolinic and kynurenic acids. Nicotinylalanine, an analogue of kynurenine, inhibited this endotoxin-induced accumulation of quinolinic acid while potentiating the accumulation of kynurenic acid. The possibility of significantly increasing brain concentrations of kynurenic acid without a concomitant increase in quinolinic acid may provide a useful approach for studying the role of these electrophysiologically active tryptophan metabolites in brain function and preventing the possible toxic actions of abnormal synthesis of quinolinic acid.     

Expression and quantification of firefly luciferase under control of Rhizobium meliloti symbiotic promoters

We have tested the use of firefly luciferase for monitoring regulated symbiotic nitrogen fixation gene expression. Broad-host-range plasmids carrying translational fusions of Rhizobium meliloti nifH, fixA and nifA promoters were constructed. Despite low levels of promoter activity the absence of Escherichia coli endogenous luminescence and the high sensitivity of the bioluminescent assay for firefly luciferase allowed rapid screening for functional luciferase expression. Plasmids containing symbiotic promoter-luc fusions were established in R. meliloti. Luciferase activity was detected and measured in both vegetative and symbiotic cells giving comparable results with those obtained by beta-galactosidase assays. In addition, the luciferase assay was quicker, more sensitive and could be carried out with unrestricted cells. Furthermore, bioluminescence was high enough in alfalfa nodules containing nifH—luc fusion to be observed by a dark-adapted eye and photographed.     

Reconstitution of sodium channels in large liposomes formed by the addition of acidic phospholipids and freeze-thaw sonication

Phosphatidylcholine (PC) alone or with phosphatidylethanolamine (PE) are sufficient for the reconstitution of Na+ channels in planar lipid bilayers. However, when Na+ channels were first reconstituted into liposomes using the freeze-thaw-sonication method, addition of acidic phospholipids, such as phosphatidylserine (PS), to the neutral phospholipids was necessary to obtain a significant toxin-modulated 22Na uptake. To further investigate the acidic phospholipid effect on reconstitution into liposomes, Na+ channels purified from Electrophorus electricus electrocytes were reconstituted into liposomes of different composition by freeze-thaw sonication and the effect of batrachotoxin and tetrodotoxin on the 22Na flux was measured. The results revealed that, under our experimental conditions, the presence of an acidic phospholipid was also necessary to obtain a significant neurotoxin-modulated 22Na influx. Though neurotoxin-modulated 22Na fluxes have been reported in proteoliposomes made with purified Na+ channels and PC alone, the 22Na fluxes were smaller than those found using lipid mixtures containing acidic phospholipids. Electron microscopy of negatively stained proteoliposomes prepared with PC, PC/PS (1:1 molar ratio), and PS revealed that the acidic phospholipid increases the size of the reconstituted proteoliposomes. The increment in size caused by the acidic phospholipid, due to the associated increase in internal volume for 22Na uptake and in area for Na+ channel incorporation, appears to be responsible for the large neurotoxin-modulated 22Na fluxes observed.     

Characterization by photoaffinity labeling of a steroid binding protein in rat liver plasma membrane

Summary The mechanism of steroid uptake by the cell remains controversial. [³H]R5020 was utilized to characterize by photoaffinity labeling the steroid binding site in plasma membrane. This binding was saturable, reversible and had one type of binding site (K _d = 33 ± 4 nm, B _max = 32 ± 2 pmol/mg). [³H]R5020 could be prevented from binding by a variety of steroids (cortisol, progesterone, deoxycorticosterone, and levonorgestrel); estradiol did not have affinity for this binding site. The kinetics of R5020 photoactivation was time dependent and saturable. SDS-PAGE showed a specific band which corresponded to a 53-kDa peptide. The sucrose density gradient analysis has revealed the existence of a protein with a sedimentation coefficient of 3.6 ± 0.2 S. This polypeptide shows different characteristics than cytosolic steroid receptor or serum steroid binding proteins. This binding protein could correspond to the steroid binding site previously found in the plasma membrane.This work was supported by grants PB85-0461 from the Comisión Asesora de Investigatión Científica y Técnica and PGV-8612 from the Departamento de Educatión, Universidades e Investigation del Gobierno Vasco. We thank Roussel-Uclaf (France) for the nonradioactive RU-steroids kindly provided.     

Cell suspension cultures ofOnonis natrix L. subspeciesramosissima: Establishment and culture conditions

Summary Explants from petioles, folioles or hypocotyls ofOnonis natrix have been used for calli initiation. Hypocotyls inoculated on MS medium supplemented with 2% sucrose and 0.5 mg.1^–1 2,4-D / 1 mg.1^–1 Kin showed to be the best primary explant. Cell suspension cultures were established in MS basal medium supplemented with 2% sucrose, 0.5 mg.1^–1 NAA or 2,4-D and 1 mg.1^–1 Kin. Different subculturing periods, inoculum density, hormonal supplementation and sucrose concentration were assayed in order to obtain the best culture growth conditions. The optimal conditions were achieved with cultures initiated with 40 g.1^–1 of initial inoculum, growing in MS basal medium supplemented with 4% sucrose, 0.5 mg.1^–1 NAA and 1 mg.1^–1 Kin subcultured every twelve days. Under these experimental conditions, the cultures showed a doubling time of 36.3 hours.     

Anoxic survival of the isolated cerebellum of the turtle Pseudemis scripta elegans

Intact turtle brain provides a useful model for the study of anoxia and potential survival strategies, since this tissue maintains transmembrane ion gradients and ATP levels during prolonged anoxia and recovers functional activity afterwards. Since isolated tissues offer experimental advantages, the present study sought to determine effects of anoxia on the isolated turtle cerebellum and to define relationships between anoxia survival and glucose supply. In normoxia, the extracellular potassium ([K+]o) activity and evoked potentials were maintained with 5 mM glucose, but 20 mM glucose was required to maintain adenosine triphosphate (ATP) levels and prevent significant increases in [K+]o during anoxia. Inhibition of glycolysis by iodoacetic acid (IAA) during anoxia provoked large increases in [K+]o at all glucose levels. These results demonstrate the usefulness of the isolated turtle cerebellum for studies of anoxic survival since this tissue can maintain ATP levels and [K+]o during prolonged anoxia with 20 mM glucose in the artificial cerebrospinal fluid medium. They also suggest the presence of a Pasteur effect at least during the transition to a hypometabolic state.