Russell E. MacDonald

[35] Light-induced transport in Halobacterium halobium

Publisher Summary This chapter discusses that study of the transport of substrates into intact bacteria is often complicated (1) by multiple sources of energy by multiple transport pathways, (2) by difficulties in characterizing the intracellular milieu, and (3) by catabolic processes that decrease the size of metabolite pools. For these reasons, envelope vesicles lacking most cytoplasmic components have been prepared from a variety of bacteria. The ease of manipulating the internal contents of such vesicles is a particularly valuable property, because transmembrane ionic gradients are recognized to play a major role in coupling metabolic energy to the accumulation of various substrates. Membranes of halobacteria contain a relatively simple H + translocation system, consisting of the protein–retinal complex called “bacteriorhodopsin,” which is activated by light. Bacteriorhodopsin molecules are located in distinct “purple membrane” patches, continuous with the cytoplasmic membrane. Transport in these vesicles can be energized either by such H + movements or by prearranged cation gradients.

Studies on the relation of thiomethyl-β-d-galactoside accumulation to thiomethyl-β-d-galactoside phosphorylation in Staphylococcus aureus HS1159☆

Abstract 1. 1. The nature of thiomethyl-β- d -galactoside (TMG) trasport and phosphorylation by Staphylococcus aureas HS1159 has been investigated. 2. 2. The enzyme system which catalyzes the production of thiomethyl-β- d -galactoside 6-phosphate (TMG-6- P ) is dependent on phosphoenolpyruvate and is similar to the phosphotransferase reported by Kundig, Ghosh and Roseman . 3. 3. The extent of uptake of TMG by S. aureus HS1159 can be altered by changes in temperature, the presence of iodoacetate, and by genetic mutation. However, in all cases where uptake is measurable, all the [ 14 C]TMG accumulated within the cells is [ 14 C]TMG-6- P . 4. 4. The suggestion by Kennedy and Scarborough that the phosphorylated sugar is the substrate of the staphylococcal ‘β-galactosidase’, taken together with the experiments presented here, lead to the hypothesis that in Staphylococcus, galactoside permeation may depend upon the first reaction of galactoside metabolism.

Partial purification and reconstitution of the aspartate transport system from Halobacterium halobium.

Membrane vesicles of Halobacterium halobium R1Wrm bind to an aspartic acid-agarose affinity column. After disruption of the bound vesicles by low ionic strength, a protein fraction is eluted from the column with 2.5% cholate in 3 M NaCl. When this fraction is reconstituted with soybean lipids to form proteoliposomes, the proteoliposomes exhibit active aspartate accumulation. Aspartate transport in the reconstituted system is driven by a chemical sodium gradient (out greater than in), exhibits sensitivity to an electrical potential, and is specific for L-aspartate. These characteristics are consistent with observations on aspartate transport in intact membrane vesicles of H. halobium. Initial aspartate transport rates in the reconstituted system are about ninefold enhanced over the native system. The system developed should be useful in future purification schemes and studies of the molecular details of membrane transport.

Enzyme crypticity as an indicator of membrane orientation in envelope vesicles from halobacteria

Summary A membrane fraction prepared by freeze-thawing envelope vesicles from Halobacterium halobium exhibits menadione-dependent NADH dehydrogenase activity (EC 1.6.9.9.3). The activity is “unmasked” by Triton X-100. Activity in freeze-thawed membrane preparations is more firmly membrane-bound than in sonicated vesicle preparations. Affinity for NADH is higher than in untreated vesicles and pyridine adenine dinucleotide specificity is the same. The data indicate that freeze-thawing causes reintegration of an adsorbed, extrinsic form of the dehydrogenase to yield an intrinsic, more native-like form of the enzyme. It is concluded that the extrinsic NADH dehydrogenase activity in vesicle preparations is not directly related to vesicle orientation.

[57]Demonstration of primary sodium transport activity in Halobacterium halobium envelope vesicles

Publisher Summary This chapter demonstrates primary sodium transport activity in Halobacterium halobium envelope vesicles. For assay of halorhodopsin activity, vesicles with di- and trinucleotides, radioactive amino acids, and various ions are used. The concentrations of the various compounds or ions loaded into the vesicles are measured and calculated using the water volume method. Two methods of monitoring the formation of membrane potential difference on illumination of M vesicles are used. These involve accumulation of membrane permeant cations: isotopically labeled [ 3 H]TPMP + or a fluorescent dye, di-O-C 5 -(3). The isotope accumulation is monitored by membrane filtration and counting techniques as done for a typical transport experiment. A second method of monitoring the formation of potential difference involves the partitioning of the fluorescent cyanine dye di-O-C 5 -(3). Internal Na + are measured directly with atomic absorption by collecting an aliquot of vesicles, washing them free of external Na + and extracting the internal Na + into water.

Photophosphorylation in cell envelope vesicles from Halobacteriumhalobium

Abstract We have prepared vesicles from cell envelope membranes of Halobacterium halobium strains R1 and ET-15 which are able to synthesize ATP in response to illumination. This photophosphorylation is inhibited by dicyclohexylcarbodiimide (DCCD) and by phloretin. ATP synthesis in L vesicles from the R1 strain (which contain bacteriorhodopsin) is inhibited by the protonophore 1799 but not by valinomycin. In M vesicles from the R1 strain and in ET-15 vesicles (both contain halorhodopsin) photophosphorylation is inhibited by both 1799 and valinomycin. These data are consistent with the idea that light-driven ATP synthesis can be coupled to the electrochemical H+ gradient generated by bacteriorhodopsin or by halorhodopsin through the membrane potential component of protonmotive force.