Isolation and purification of bacterial membrane proteins by the use of organic solvents: The lactose permease and the carbodiimide-reactive protein of the adenosinetriphosphatase complex of escherichia coli

Techniques for the solubilization and fractionation of integral membrane proteins have been developed in recent years. A small portion of membrane protein (about 2%, proteolipid fraction) will partition into chloroform or 1-butanol, and, in several cases, these proteins retain functional activity. A virtually complete solubilization can be achieved at neutral pH by use of aprotic solvents, like hexamethylphosphoric triamide or N-methylpyrrolidone. At relatively low concentrations (< 3 M) aprotic solvents inhibited β-D-galactoside transport by whole cells and the derivative membrane vesicles of Escherichia coli, but this inhibition could be largely reversed by a simple washing procedure. At higher concentrations of aprotic solvent (5–6 M), 50–80% of the total protein of lactose transport-positive membrane vesicles was solubilized. When these extracts were added to intact lactose transport-negative membrane vesicles, lactose transport was reconstituted, the required energy being provided by either respiration (e.g., addition of D-lactate) or by a K⁺ diffusion potential established with the aid of valinomycin. The dicyclohexylcarbodiimide (DCCD)-reactive subunit of the E. coli ATPase complex was found to partition into chloroform, and to be amenable to further purification in organic solvent. Ether precipitation and chromatography on DEAE-cellulose and hydroxypropyl-Sephadex G-50 yielded an homogeneous polypeptide of an apparent molecular weight of 9,000. The purified and unlabeled DCCD-reactive protein was incorporated into K⁺-loaded liposomes, and a membrane potential was generated by the addition of valinomycin. There are indications that the DCCD-reactive protein alone made the membrane specifically permeable for protons.     

Deciphering the “Fuzzy” Interaction of FG Nucleoporins and Transport Factors Using Small-Angle Neutron Scattering

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Molecular responses of plants to an increased incidence of heat shock

Abstract. Climatic change as a result of the greenhouse effect is widely predicted to increase mean temperatures globally and, in turn, increase the frequency with which plants are exposed to heat shock conditions, particularly in the semi-arid tropics. The consequences of extreme high-temperature treatments on plants have been considered, particularly in relation to the synthesis of heat shock proteins (HSPs) and the capacity to acquire thermotolerance. The heat shock response is described using results obtained with seedlings of the tropical cereals, sorghum ( Sorghum bicolor ) and pearl millet ( Pennisetum glaucum ). A gradual temperature increase, as would occur in the field, is sufficient to induce thermotolerance. The synthesis of HSPs is a transient phenomenon and ceases once the stress is released. Despite the persistence of the HSPs themselves, de novo synthesis of HSPs is required for the induction of thermotolerance each time high temperatures are encountered. The effect of a repeated, diurnal heat shock was investigated and genotypic differences found in the ability to induce the heat shock response repeatedly.     

XylS domain interactions can be deduced from intraallelic dominance in double mutants of Pseudomonas putida.

Summary The XylS protein is the positive regulator of the TOL plasmid-encoded meta-cleavage pathway for the metabolism of alkylbenzoates in Pseudomonas putida. This protein is activated by a variety of benzoate analogues. To elucidate the functional domains of the regulator and their interactions, several fusions of the XylS C-terminus to MS2 polymerase and of the N-terminus to -galactosidase were constructed but all are inactive. In addition, 15 double mutant xylS genes were constructed in vitro by fusing parts of various mutant genes to produce mutant regulators exhibiting C-terminal and N-terminal amino acid substitutions. The phenotypic properties of the parental single mutant genes, and those of the double mutant genes, suggest that the C-terminal region is involved in binding to DNA sequences at the promoter of the meta-cleavage pathway operon, and that the benzoate effector binding pocket includes critical residues present at both the N-terminal and C-terminal ends of the protein. The intraallelic dominance of the Ile229 (Ser229 Ile) and Val274 (Asp274 Val) substitutions over the N-terminal His4l (Arg4l His) substitution, and the intraallelic dominance of Thr45 (Arg45 Thr) over Ile229 and Val274, support the proposal that these two regions of the regulator interact functionally. Combination of the Leu88 (Trp88 Leu) and Arg256 (Pro256 Arg) substitutions did not suppress the semiconstitutive phenotype conferred by Leu88, but resulted in a protein with altered ability to recognize benzoates. In contrast, the Leu88 semiconstitutive phenotype was suppressed by Va1288 (Asp288 Val), and the double mutant was susceptible to activation by benzoates. The results suggest that intramolecular interactions between the C- and N-terminal regions of XylS are critical for activation of the regulator by the effector.     

Immigration,remittances, and transnational social capital formation: a Cuban case study

Social capital takes on distinctive meaning in the context of large-scale immigration from poor to rich countries. In this article, characteristics of social capital embedded in transnational networks and norms, conditions conducive to the formation of such networks, and effects such networks have are extrapolated from an analysis of how and why cross-border relations among Cuban-Americans and Cubans in their homeland have changed since the 1959 Castro-led revolution. The transnational social capital generating benefit on which the study focuses is remittances. Remittances are of growing global importance to less developed countries, and in some countries they generate more revenue than foreign aid and foreign investment. The analysis addresses a range of unintended as well as intended consequences remittances may have.     

Oxidative Modification of Proteins: Oxidation of Tryptophan and Production of Dityrosine in Purified Proteins Using Fenton's System

Specific features of metal-catalyzed oxidation (MCO) of purified proteins (human serum albumin and human erythrocyte superoxide dismutase) were analyzed by the oxidation level of tryptophan and tyrosine. The production of dityrosine cross-links and the oxidation of tryptophan residues were recorded by fluorescence. The degree of oxidative modification of the amino acid residues of the proteins depended on the concentration of the Fenton's medium components and on the incubation time. These changes were different in different proteins. By electrophoresis and gel-permeation chromatography, changes in the superoxide dismutase structure are shown to be caused by oxidative modification of the enzyme and to be accompanied by a decrease in its activity. Findings with OH^. scavengers (mannitol and ethanol) suggest that oxidative modification of the proteins in Fenton's medium should be associated not only with hydroxyl radical but also with ferryl and perferryl ions and with the radical PH₃.