Transport in halobacterium halobium: Light-induced cation-gradients, amino acid transport kinetics, and properties of transport carriers
✍ Scribed by Lanyi, Janos K.
- Publisher
- Wiley (John Wiley & Sons)
- Year
- 1977
- Tongue
- English
- Weight
- 538 KB
- Volume
- 6
- Category
- Article
- ISSN
- 0091-7419
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✦ Synopsis
Abstract
Cell envelope vesicles prepared from H. halobium contain bacteriorhodopsin and upon illumination protons are ejected. Coupled to the proton motive force is the efflux of Na^+^. Measurements of ^22^Na flux, exterior pH change, and membrane potential, ΔΨ (with the dye 3,3′‐dipentyloxadicarbocyanine) indicate that the means of Na^+^ transport is sodium/proton exchange. The kinetics of the pH changes and other evidence suggests that the antiport is electrogenic (H^+^/Na^+^ > 1). The resulting large chemical gradient for Na^+^ (outside > inside), as well as the membrane potential, will drive the transport of 18 amino acids. The 19th, glutamate, is unique in that its accumulation is indifferent to ΔΨ: this amino acid is transported only when a chemical gradient for Na^+^ is present. Thus, when more and more NaCl is included in the vesicles glutamate transport proceeds with longer and longer lags. After illumination the gradient of H^+^ collapses within 1 min, while the large Na^+^ gradient and glutamate transporting activity persists for 10–15 min, indicating that proton motive force is not necessary for transport. A chemical gradient of Na^+^, arranged by suspending vesicles loaded with KCl in NaCl, drives glutamate transport in the dark without other sources of energy, with V~max~ and K~m~ comparable to light‐induced transport. These and other lines of evidence suggest that the transport of glutamate is facilitated by symport with Na^+^, in an electrically neutral fashion, so that only the chemical component of the Na^+^ gradient is a driving force.
The transport of all amino acids but glutamate is bidirectional. Actively driven efflux can be obtained with reversed Na^+^ gradients (inside > outside), and passive efflux is considerably enhanced by intravesicle Na^+^. These results suggest that the transport carriers are functionally symmetrical. On the other hand, noncompetitive inhibition of transport by cysteine (a specific inhibitor of several of the carriers) is only obtained from the vesicle exterior and only for influx: these results suggest that in some respects the carriers are asymmetrical.
A protein fraction which binds glutamate has been found in cholate‐solubilized H. halobium membranes, with an apparent molecular weight of 50,000. When this fraction (but not the others eluted from an Agarose column) is reconstituted with soybean lipids to yield lipoprotein vesicles, facilitated transport activity is regained. Neither binding nor reconstituted transport depend on the presence of Na^+^. The kinetics of the transport and of the competitive inhibition by glutamate analogs suggest that the protein fraction responsible is derived from the intact transport system.