Respiration-Driven Na+-Pump in Alkaliphilic Bacillus S1-3
Akinobu OSHIMA*, Naohisa YOSHIOKA, and Tetsuo ONODA
Department of Biological Science, Shimane University, 1060 Nishikawatsu-cho, Matsue 690, Japan
It has been reported that primary Na+-pumps found in marine bacteria enable to generate ΔμNa+ when they cannot produce sufficient ΔμH+. Therefore, alkaliphilic bacteria, producing less amount of ΔμH+ under the optimal growing conditions, appear to have a primary Na+-pump, like that observed in marine bacteria. In order to explore the character of a primary Na+-pump of alkaliphlic bacteria, we prepared everted membrane vesicles from alkaliphilic bacterial strain Bacillus S1-3 to measure the activity of several key enzymes. It was shown that the NADH oxidase activity was increased when Na+ was added in reaction mixtures and the optimal activity was observed at 40 mM of Na+. In the case of K+, it gave relatively smaller effect on the elevation of enzyme activity than Na+. It has been already known that bacteria that Na+ enhances NADH oxidase activity possess the respiration-driven primary Na+-pump. The activity of NADH dehydorgenase was slightly increased by the additon of 40 mM of Na+ or K+ and the presence of Na+ was not absolutely required for its activity. The activity of cytochrome oxidase was dependent on some monovalent cations. But, Na+ did not result in any drastic increment on its activity. Finally, NAD: menaquinone (MK) reductase was strongly activated by the presence of Na+, while slightly activated by the presence of K+. These results are suggesting that a Na+-dependent activation site in NADH oxdase is integrated into the NADH: MK reductase segment. Interestingly, the activity of NA: MK reductase of the alkaliphile was not inhibited by 2-n-heptyl-4-hydoxyquinone (HQNO), although NAD: quinone reductase of marine bacteria was strongly inhibited by HQNO.
We also determine a membrane potential and its sensitivity to protonophore and Na+-ionophore as described by Unemoto et al (1), to examine the presence of respiration driven Na+--pump. The membrane potential generated by NADH oxidation still remained after addition of the protonophore 3, 3', 4, 5'-tetrachlorosalicylanide (TCS) to reaction mixtures, but successive addition of Na+-conducting ionophore monensin resulted in complete dissipation of the potential. The membrane potential insensitive to protonophore was detected at the concentration of less than 20 μM of TCS. In contast with this, the membrane potential generated by succinate oxidation was lower, which was completely disappeared by the addition of TCS.
In conclusion, our data suggest that not only H+ but also Na+ is extruded during the oxidation of NADH at the respiratory chain of alkaliphilic Bacillus, where a primary Na+-pump is coupled to the NAD: MK reducatase segment.
1. Unemoto, T., Akagawa, A., Mizugaki, M., and Hayashi, M. (1992) J. Gen. Microbiol., 138, 1999-2005.
