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R Renthal

Publications and source records attributed to R Renthal.

29 records · Page 2Linked to original sources
PubMed1983

A cleavable cross-linking reaction for protein carboxyl groups.

L-(+)-tartaric acid dihydrazide was coupled to the purple membrane from Halobacterium halobium with a water soluble carbodiimide. Gel electrophoresis analysis of the product revealed the formation of bacteriorhodopsin dimers, trimers and higher polymers. Most of the cross-links were removed by treatment with papain, demonstrating involvement of the carboxyl-rich carboxyl-terminal region of bacteriorhodopsin in the reaction. The cross-links were cleavable by periodate oxidation.

Bacteriorhodopsins↗
PubMed1981

Light-induced changes in H+ binding to the purple membrane. Effect of pH, light, temperature, and ionic strength.

Under continuous illumination, isolated planar sheets of purple membrane from Halobacterium halobium acidify the surroundings at alkaline pH. This light-induced change in H+ binding to the purple membrane (delta h) was studied by differential titration under varying conditions of pH, temperature, ionic strength, salt composition, light intensity, and wavelength. A maximum acidification was found between pH 9 and 10, with delta h less at neutral or more alkaline pH, consistent with a previously proposed three-state model. The light intensity and wavelength dependence also support this model. The temperature dependence of delta h, interpreted in terms of the three-state model, is anomalous. The apparent enthalpy of proton dissociation (delta H0) is -6 kcal/mol, a value of opposite sign to the expected delta H0 for a group of pK = 10. The apparent activation energy (Ea) for proton uptake is 14 kcal/mol in 15 mM NaCl and 18 kcal/mol in 3 M KCl, 5 to 10 times too large for a diffusion-limited proton transfer reaction from water. However, both delta H0 and Ea are consistent with conformational changes linked to light-independent proton dissociation and pump-dependent proton uptake. An increase in ionic strength increases delta h. This effect is shown to be quantitatively explained by a high negative electrostatic surface potential, which accumulates protons in a diffuse electrical double layer.

Bacteriorhodopsins↗
PubMed1980

Carbodiimides inhibit the acid-induced purple-to-blue transition of bacteriorhodopsin.

Reaction of purple membrane with water soluble carbodiimides inhibits the spectral transition from purple to blue observed at acid pH. The pK and Hill constant for this transition are shifted from 3.4 to 2.6 and from 1.8 to 0.85, respectively. The results suggest a connection between the uptake side of the proton pump and the purple-to-blue transition.

Bacteriorhodopsins↗
PubMed1979

Reaction of the purple membrane with a carbodiimide.

Purple membrane was reacted with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at pH 4.5 and 8.0. At pH 4.5, the reaction yields cross-linked bacteriorhodopsin. The cross-linking is inhibited by pretreatment of the membrane with papain, or by the presence of carbohydrazide or glycine ethyl ester in the reaction mixture. The product of the pH 8.0 reaction is not cross-linked, but it displays altered properties. Two measures of photochemical activity (light-induced change in proton binding (delta h) and decay of photointermediate M) show changes indicative of slowed proton uptake. The delta h is increased by ethyl dimethylaminopropylcarbodiimide. This increase is unaffected by pretreatment of the membrane with papain, and it is not reversed by NH2OH. However, the reaction is inhibited by millimolar concentrations of CaCl2. The altered delta h is not apparent in detergent-solubilized membranes. Ethyl dimethylaminopropylcarbodiimide does not appear to cause a large alteration in the membrane surface charge, as measured by Ca2+ binding. We conclude that (1) at acid pH, ethyl dimethylaminopropylcarbodiimide can be used for cross-linking or for attachment of specific probes to the C-terminal region of bacteriorhodopsin, and hence to the cytoplasmic side of the purple membrane, and (2) at alkaline pH, ethyl dimethylaminopropylcarbodiimide reacts at a diffent type of site and appears to inhibit the proton pump.

Bacteriorhodopsins↗
PubMed1976

Light-induced membrane potential and pH gradient in Halobacterium halobium envelope vesicles.

Illumination of envelope vesicles prepared from Halobacterium halobium cells causes translocation of protons from inside to outside, due to the light-induced cycling of bacteriorhodopsin. This process results in a pH gradient across the membranes, an electrical potential, and the movements of K+ and Na+. The electrical potential was estimated by following the fluorescence of a cyanine dye, 3,3'-dipentyloxadicarbocyanine. Illumination of H. halobium vesicles resulted in a rapid, reversible decrease of the dye fluorescence, by as much as 35%. This effect was not seen in nonvesicular patches of purple membrane. Observation of maximal fluorescence decreases upon ilumination of vesicles required an optimal dye/membrane protein ratio. The pH optimum for the lightinduced fluorescence decrease was 6.0. The decrease was linear with actinic light intensity up to about 4 X 10(5) ergs cn-2 s-1. Valinomycin, gramicidin, and triphenylmethylphosphonium ion all abolished the fluorescence changes. However, the light-induced pH change was enhanced by these agents. Conversely, buffered vesicles showed no pH change but gave the same or larger fluorescence changes. Thus, we have identified the fluorescence decrease with a light-induced membrane potential, inside negative. By using valinomycin-K+-induced membrane potentials, we calibrated the fluorescence decrease with calculated Nernst diffusion potentials. We found a linear dependence between potential and fluorescence decrease of 3 mV/%, up to 90 mV. When the envelope vesicles were illuminated, the total proton-motive force generated was dependent on the presence of Na+ and K+ and their concentration gradients across the membrane. In general, K+ appeared to be more permeable than Na+ and, thus, permitted development of greater pH gradients and lower electrical potentials. By calculating the total proton-motive force from the sum of the pH and potential terms, we found that the vesicles can produce proton-motive forces near--200 mV.

Cell Membrane↗
PubMed1976

Light-induced glutamate transport in Halobacterium halobium envelope vesicles. II. Evidence that the driving force is a light-dependent sodium gradient.

Illumination of cell envelope vesicles from H. halobium causes the development of protonmotive force and energizes the uphill transport of glutamate. Although the uncoupler, p-trifluoromethoxycarbonyl cyanide phenylhydrazone (FCCP), and the membrane-permeant cation, triphenylmethylphosphonium (TPMP+), are inhibitory to the effect of light, the time course and kinetics of the production of the energized state for transport, and its rate of decay after illumination, are inconsistent with the idea that glutamate accumulation is driven directly by the protonmotive force. Similarities between the light-induced transport and the Na+-gradient-induced transport of glutamate in these vesicles suggest that the energized state for the amino acid uptake in both cases consists of a transmembrane Na+ gradient (Na+out/Na+in greater than 1). Rapid efflux of 22Na from the envelope vesicles is induced by illumination. FCCP and TPMP+ inhibit the light-induced efflux of Na+ but accelerate the post-illumination relaxation of the Na+ gradient created, suggesting electrogenic antiport of Na+ with another cation, or electrogenic symport with an anion. The light-induced protonmotive force in the H. halobium cell envelope vesicles is thus coupled to Na+ efflux and thereby indirectly to glutamate uptake as well.

Biological Transport, Active↗