Protons In Response Research Articles

Structure-function studies on bacteriorhodopsin. V. Effects of amino acid substitutions in the putative helix F.

To test structural and mechanistic proposals about bacteriorhodopsin, a series of analogues with single amino acid substitutions has been studied. Mutants in the proposed helix F of bacteriorhodopsin were chosen for investigation because of the probable interaction of this part of the protein with the retinal chromophore. Seven mutants of the bacteriorhodopsin gene were constructed by site-directed mutagenesis, and the gene products were expressed in Escherichia coli. The resulting mutant proteins were purified and assayed for their ability to interact with retinal in phospholipid/detergent micelles to form a bacteriorhodopsin-like chromophore. Four mutants, Ser-183----Ala, Tyr-185----Phe, Ser-193----Ala, and Glu-194----Gln, bound retinal to give pigments with absorption maxima approximately the same as the wild type. Three mutant opsins bound retinal to give chromophores that were blue-shifted relative to the wild type. Two Trp----Phe substitutions at positions 182 and 189 gave absorption maxima of 480 and 524 nm, respectively, and the mutant Pro-186----Leu gave a pigment with an absorption maximum of 470 nm. However, none of the amino acid substitutions eliminated the ability of the mutant bacteriorhodopsin to pump protons in response to illumination.

Iron respiration-driven proton translocation in aerobic bacteria.

Washed cell suspensions of Aquaspirillum magnetotacticum MS-1, A. itersonii E12639, Bacillus subtilis 6633, and Escherichia coli CSH27 translocated protons in response to the added oxidant O2 or NO3-, with triphenylmethylphosphonium bromide as the permeant ion. Iron respiration-driven proton translocation was observed in A. magnetotacticum MS-1, B. subtilis, and E. coli but not in a nonmagnetic strain of A. magnetotacticum (strain NM-1A) or with A. itersonii. Proton translocation to Fe3+ was totally inhibited by 500 microM NaN3 or 0.5 microM carbonyl cyanide m-chlorophenylhydrazone.

Spectroscopic and functional properties of subunit III-depleted cytochrome oxidase.

Beef heart cytochrome c oxidase has been depleted of subunit III by treatment with chymotrypsin. The removal of subunit III has been evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel fluorography of preparations of the oxidase labeled with [14C]dicyclohexylcarbodiimide prior to proteolysis. Removal of subunit III resulted in a perturbation of the visible spectrum of reduced cytochrome oxidase. Subunit III-depleted oxidase is spectroscopically very similar to the oxidase from Paracoccus denitrificans. When reconstituted into liposomes, the depleted enzyme still pumped protons in response to a pulse of reduced cytochrome c. The H+/e- stoichiometry averaged 0.5. Redox-linked proton translocation could be observed only when respiratory control ratios were higher than 3 and the reductant pulse was of a magnitude that allowed for no more than 5 turnovers of the oxidase.

Extensor and Flexor Protoplasts from Samanea Pulvini

Protoplasts were isolated from extensor and flexor regions of open pulvini of the nyctinastic tree Samanea saman. Both types of protoplasts undergo many changes during isolation. Extensor protoplasts are univacuolate in vivo, but some become multivacuolate. All flexor protoplasts are univacuolate. In an open pulvinus, extensor cells have a higher osmotic pressure than flexor cells. However, both types of protoplasts can be isolated with optimal yield using the same osmoticum (0.5 molar sorbitol) in the digestion medium. This suggests that some leakage of osmoticum occurs during harvest or digestion, especially from extensor tissue. Despite these changes, both types of protoplasts extrude protons in response to 10 micromolar fusicoccin (1.6-1.8 nanoequivalent/10(6) protoplasts/minute), demonstrating that the protoplasts are metabolically active and that proton transport mechanisms must be at least partially functional. The changes in vacuolar structure and osmotic pressure are what one might expect if the protoplasts, which are isolated from open pulvini, take on characteristics of cells in a closed pulvinus.

Effect of Salt on Auxin-Induced Acidification and Growth by Pea Internode Sections

The capacity of excised internode sections of pea to grow and secrete protons in response to indoleacetic acid (IAA) and Ca(2+) and K(+) treatments was examined. By incubating unpeeled and unabraded sections in rapidly flowing solutions, it was shown that acidification of the external medium in the presence or absence of IAA is dependent on the presence of Ca(2+) and K(+). Similar results were obtained when unpeeled and unabraded sections were incubated in dishes with shaking. When peeled or abraded sections were incubated with shaking in IAA, H(+) release was also dependent on the presence of Ca(2+) and K(+). The release of H(+) from sections incubated in Ca(2+) and K(+) is not caused by displacement of H(+) from binding sites in the cell wall. Rather, the release of protons from sections is temperature dependent, and it is concluded that this is a metabolically linked process. Although Ca(2+) and K(+) are essential for the release of H(+) from isolated stem sections of peas, these cations do not influence elongation. Despite the large increase in proton release induced by Ca(2+) and K(+) either in the presence or absence of auxin, growth in the presence of these ions was never greater than it was in their absence. Furthermore, cations do not affect the neutral sugar or uronic acid composition of the solution which can be centrifuged from isolated sections. As is the case for growth, an increase in the neutral sugar and uronide composition of the cell wall solution is dependent only on IAA. It is concluded that IAA-induced growth of pea stem sections is independent of the secretion of protons.

Sodium ion-proton antiport in a marine bacterium.

Alteromonas haloplanktis ejected protons in response to a brief respiratory pulse; the rate of decay of the resulting pH change was accelerated when Na+ was present in the suspension medium. The addition of an anaerobic NaCl solution to an essentially Na+-free anaerobic bacterial suspension induced the acidification of the suspension medium. These results and others discussed provide substantial evidence for the existence of an Na+-H+ antiporter in this organism.