Volume changes in illuminated cell envelope vesicles, prepared from various
Halobacterium halobium strains, were measured with an ESR method. We demonstrated light-dependent swelling of vesicles ...which contained halorhodopsin (an inward-directed light-driven chloride pump), and shrinking of vesicles which contained bacteriorhodopsin (an outward-directed light-driven proton pump coupled to a proton/sodium antiporter). The swelling of the halorhodopsin vesicles was not inhibited by uncouplers or gramicidin, but the shrinking of the bacteriorhodopsin-vesicles was abolished by these ionophores. These findings confirm earlier models for ion circulation in these systems. Vesicles from strains which contained both pigments showed relatively small net volume changes upon illumination. A scheme of ionic transport in
H. halobium cells is suggested, in which the inward movement of K
+ exceeds the outward movement of Na
+, and the difference equals the Cl
− uptake, so as to provide the net gain of KCl necessary for volume increases during cell growth.
The concept of osmotic regulation in plant cells, including both turgor pressure regulation by transport of inorganic ions into giant-celled algae and the accumulation of organic compounds in several ...microscopic algae and tissue cells is critically investigated. The importance of the cellular water activity and water structure and their influence on the solubility and hydration sphere of the biopolymers is strengthened.
The hyperosmotic conditions, causing the synthesis of polyols or proline in several plant cells, include the state of plasmolysis. Under these conditions, a reduction of the cellular water activity is assumed to impair the hydration sphere of the cellular constituents. The function of the polyols and proline is interpreted to improve the solubility of the cellular constituents. Until now, there is not suficient experimental evidence that the accumulated organic compounds act by an osmotic balance. During turgor pressure regulation in giant-celled luarine algae plasmolysis is not involved. It is suggested that changes of sea water salinities, occurring under the natural condition and inducing the corresponding alterations of the internal osmotic pressure, do not impair the hydration of the cytoplasmic components. Probably they are adapted to a certain range of water activities by a different structure. Therefore, the solubility-improving mechanism with organic compounds is not necessary, but the maintenance of turgor pressure is the aim of this regulatory mechanism.
These considerations led to the conclusion that the concept of an osmotic regulation is only true for turgor pressure regulation in giant algal cells.
The rate of NADH oxidation by inverted membrane vesicles prepared from the halotolerant bacterium Ba1 of the Dead Sea is increased specifically by sodium ions, as observed earlier in whole cells. The ...site of this sodium effect is identified as the NADH: quinone oxidoreductase, similarly to the other such system known, Vibrio alginolyticus (H. Tokuda and T. Unemoto (1984) J. Biol. Chem. 259, 7785-7790). Sodium accelerates quinone reduction severalfold, but oxidation of the quinol, with oxygen as terminal electron acceptor, is unaffected. The sodium-dependent pathway of quinone reduction exhibits higher apparent affinity to extraneous quinone (Q-2) than the sodium-insensitive pathway, and is specifically inhibited by 2-heptyl-4-hydroxyquinoline N-oxide. ESR spectra of the membranes contain a feature at g = 1.98 which is tentatively identified as one originating from semiquinone. This feature is increased by NADH and decreased by addition of Na+, suggesting that, as proposed from different kinds of evidence for the V. alginolyticus system, sodium affects the semiquinone reduction step. As in the other system, the site of sodium stimulation in Ba1 probably corresponds to the site of sodium translocation, which was shown earlier (S. Ken-Dror, R. Shnaiderman, and Y. Avi-Dor (1984) Arch. Biochem. Biophys. 229, 640-649) to be linked directly to a redox reaction in the respiratory chain.
Nucleotide-binding sites of the ATPase from the halophilic archaebacterium Halobacterium saccharovorum were labeled by ultraviolet irradiation in the presence of alpha-32PATP. A high-affinity site, ...located on subunit I (98 kDa), was identified as catalytic by the following criteria: ATP bound to subunit I was hydrolyzed and the cross-linked nucleotide was ADP; the specificity for ATP or ADP compared to that of other nucleotides was high; the tightly bound radionucleotide was exchangeable in the presence of excess unlabeled ATP and Mg2+; photolabeling of this site and enzyme inhibition due to tightly bound ADP were both dependent on the presence of Mg2+ and showed identical Kd values; treatment that restored the activity of the ADP-inhibited enzyme also led to the release of the tightly bound nucleotide from subunit I. In addition, a non-catalytic nucleotide-binding site was found, located on subunit II (71 kDa). This site did not hydrolyze ATP, its occupation was Mg2+ independent and the affinity for ATP and the nucleotide specificity were much lower than that of subunit I. We suspect that this site is nonspecific. These results indicate that H. saccharovorum ATPase is different from F1-ATPases which contain the catalytic site on the second largest subunit, but may be similar to other archaebacterial and vacuolar ATPases.
Full text
Available for:
BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
Exhaustive reaction with phenylglyoxal removed 9 of the 12 arginine and 1 of the 2 lysine residues in detergent-solubilized halorhodopsin, without affecting the chromophore. The consequences of this ...extensive removal of positive charges on various chloride-binding equilibria and the photochemistry were evaluated. No significant effects were seen on the affinity of Site I to chloride and on the increase in the pKa of Schiff-base deprotonation, which is caused by the chloride binding at this site. No significant effects were seen on the affinity of Site II to chloride, either. However, the photocycle of the pigment was affected. Kinetic modeling of the observed changes in flash-induced absorption changes suggests that the modification increases the affinity of the main halorhodopsin photointermediate to chloride by about fourfold. If chloride translocation involves release of chloride from this intermediate during the transport cycle, the result might explain the observed partial inhibitory effects on chloride transport. Plausible models of chloride translocation include reversible binding of the anion by positively charged groups, strategically arranged in the protein. The results indicate that two of the three spectroscopically observable chloride-dependent equilibria do not depend on a large number of positively charged residues in the protein. To the extent that the unaffected equilibria represent association and dissociation which occur during chloride translocation, at least part of the chloride translocation might be accomplished with the participation of only a few positively charged residues.
In a light-dependent reaction (3.5 kilolux) at pH 5, the evolution of hexanal, ethane, and ethylene has been established with cell suspensions of the diatom, Phaeodactylum tricornutum. During this ...process, chlorophyll and carotenoids are partially bleached. Addition of 25 millimolar α-linolenic acid or 12 millimolar docosahexaenoic acid yield total pigment destruction and enhancement of ethylene and ethane formation (by about 150 and 7,600%, respectively), whereas hexanal production decreases by 70%. Eicosapentaenoic acid, the major polyunsaturated fatty acid in diatoms, stimulates both ethane and hexanal formation (by about 1,400 and 130%, respectively), but reduces ethylene production (by about 60%). This competition suggests that the production of the volatile compounds is closely connected, although hexanal and ethylene obviously possess different unsaturated fatty acids as precursors. Both the kind of the fatty acids and their relative amounts seem to determine the pattern of the evolved hydrocarbons. The presence of 10 millimolar propylgallate inhibits the evolution of the volatile compounds by about 80%, indicating that radical formation might play a key role in this light-dependent cascade of reactions.
Full text
Available for:
BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
PDF
