Naoyuki Murakami

Simultaneous determination of membrane potential and pH gradient by photodiode array spectroscopy

Membrane potential (delta psi) and pH difference (delta pH) were simultaneously determined in liposomes using a photodiode array spectrophotometer. By the use of a cyanine dye (DiS-C3(5)) and 9-aminoacridine for delta psi and delta pH probes, respectively, both changes of delta psi and delta pH could be successfully determined by photodiode array spectrometry. Each dye did not disturb the fluorescence spectrum of the other probe when its concentration was lower than 5 microM. The K+-diffusion potential-driven, FCCP(protonophore)-mediated H+-influx process in the K+-loaded liposomes was analyzed by this method. Results indicate that the kinetic behavior of H+ influx changes at a FCCP concentration of approx. 30 nM. The rate of delta pH formation increased quantitatively with increasing concentrations of FCCP up to 30 nM, but was markedly enhanced at higher concentrations, although the maximal delta pH attained was about 3 pH units in any case when a K+-diffusion potential of -180 mV was applied.

Δψ-dependent gating of Na+/H+ exchange in Halobacterium halobium: a Δg̃mH+-driven Na+ pump

Na+/H+ antiporter‐mediated 22Na+ transport was studied in envelope vesicles from Halobacterium halobium by manipulating the size of each Δg̃m+ component, ΔpH and Δψ, in the dark. Neither inside alkaline ΔpH nor outwardly directed ΔpNa+, nor a combination could facilitate 22Na+ extrusion from the vesicles. Likewise, Δψ up to 144 mV (inside negative) was not capable of initiating 22Na+ extrusion unless ΔpH existed. This extrusion was facilitated only when approx. 100 mV Δψ (gating potential) was superimposed on ΔpH (either 1 or 2). On the other hand, no uptake of 22Na+ took place even when both inside acidic ΔpH and inwardly directed Na+ gradient were imposed with or without Δψ. Under these conditions, monensin mediated the rapid uptake of 22Na+. The present results indicate that halobacterial Na+/H+ exchange is regulated not only by a Δψ‐dependent gate but also by a certain mechanism to restrict the back flux of Na+, this making the antiporter capable of functioning as an efficient Δg̃mH+‐driven pump for Na+ in a high saline environment.

Detection of two DCCD-binding components in the envelope membrane of H. halobium

From labeling studies using [14C]DCCD, the presence of two distinct DCCD‐binding sites was proved in halobacterial membrane, whose binding constants are 0.03 and 0.08 nmol DCCD/mg envelope protein, respectively. HPLC and SDS—PAGE analysis revealed that the radioactivity bound to these sites migrated with apparent molecular masses of 45 and 10.2 kDa protein components, respectively.

Cooperative regulation of the Na+H+-antiporter in Halobacterium halobium by ΔpH and Δφ

Abstract The contributions of the transmembrane pH gradient (ΔpH) and electrical potential (Δφ) to the Δ \ gm H + -driven Na+ efflux (mediated by the N,N′-dicyclohexylcarbodiimide-sensitive Na + H + -antiporter) were investigated in membrane vesicles of Halobacterium halobium. Kinetic analysis in the dark revealed that two different Na+-binding sites are located asymmetrically across the membrane: One, accessible from the external medium, has a Kd (half-maximal stimulation of Na+ efflux) of about m , and the Na+ binding to the site is a prerequisite for the antiporter activation by Δ \ gm H + . The other cytoplasmic site is the Na+ transport site. The Km for the cytoplasmic Na+ decreased as the ΔpH increased, while the Vmax remained essentially constant in the presence of defined Δφ (140 mV). On the other hand, Δφ elevation above the gating potential (~100 mV) increased the Vmax without changes in the Km in the presence of a fixed ΔpH. It was also noted that the Km value in the absence of Δφ was completely different from and far higher than that observed in the presence of Δφ (>100 mV), indicating the existence of two distinct conformations in the antiporter, resting and Δφ gated; the latter state may be reactive only to ΔpH. On the basis of the present data and the previous data on the pH effect ( N. Murakami and T. Konishi, 1989 Arch. Biochem. Biophys. 271, 515–523), a model for the ΔpH-Δφ regulation of the antiporter activation is proposed.

Functional comparison of DCCD-sensitive Na+/H+ antiporter in Halobacterium halobium with monensin

Membrane vesicles from Halobacterium halobium create a large, inside negative membrane potential (delta psi) and small, inside alkaline pH gradient (delta pH) by illumination in 3 M NaCl. delta psi was the major component of a proton electrochemical potential (delta microH+) over a pH range from 5 to 8. After DCCD treatment of the vesicles, delta psi was replaced by delta pH due to the inhibition of the intrinsic delta pH----delta psi transformation process: delta psi formation in light is markedly retarded and an inversely large delta pH is established at these pHs. DCCD-caused changes in delta psi and delta pH were completely restored to the control level by the addition of monensin, an electroneutral Na+/H+ exchanger. The ratio of DCCD-caused change in delta pH and delta psi was identical to that of monensin-recovered delta psi and delta pH. The delta psi/delta pH ratio was approximately 0.8, that is, 100 mV of delta pH was transformed into 78 mV of delta psi. The present results indicate that the intrinsic activity of the DCCD-sensitive delta pH----delta psi transformation is mediated by an electroneutral Na+/H+ exchange.

Δ\̃gmH+Δ\̃gmNa+ exchange ratio in the halobacterial Na+/H+-antiporter determined by a simultaneous measurement of ΔpH, ΔΨ and ΔpNa

Due to the difficulty in quantitative treatment of the three thermodynamic parameters involved in the Na+/H+-exchange process (the concentration differences of H+ and Na+ across the membrane (ΔpH and ΔpNa)) and the membrane potential (ΔΨ)), an accurate exchange ratio of proton electrochemical potential (Δ\gmH+) to sodium electrochemical potential (Δ\gmNa+) in the halobacterial Na+/H+-antiporter has not yet been established. In the present study, ΔpH, ΔpNa and ΔΨ were determined in the membrane vesicles kept in a steady state of illumination using [14C]salicylate, [3H]tetraphenyl phosphonium (TPP+) and 22Na serving as a ΔpH-, ΔΨ- and ΔpNa-probe, respectively, using triple-tracer liquid scintillation counting. Na+/H+-antiporter activity in the vesicle membrane was manipulated by treating the vesicle with elevated concentrations of N,N′-dicyclohexylcarbodiimide (DCCD), a specific inhibitor for the halobacterial Na+/H+-antiporter. From the comparison of DCCD-dependent fractional changes of Δ\gmH+ and Δ\gmNa+ in the light-induced overall electrochemical potential, the Δ\gmH+Δ\gmNa+-exchange ratio of 1 was deduced for the halobacterial Na+/H+-antiporter.

Application of simultaneous determination of 3H, 14C, and 22Na by liquid scintillation counting to the measurement of cellular lon-transport

A liquid scintillation counting method for simultaneous determination of three radioactive nuclides (3H, 14C, and 22Na) of biological interest was studied. By comparing the beta spectra of the three nuclides, their counting energy ranges, A, B, and C, were determined. 22NA was set high enough to avoid any spillover counts from lower-energy nuclides. Region A for 3H was set to maximize the counting efficiency. A good correlation between the counting efficiency for 22Na in region C and the counting efficiency of other nuclides in all regions was obtained. Prior to 3H and 14C dpm calculations, the 22Na counts spilled down in regions A and B were subtracted from the total counts in regions A and B. A simple linear equation was then used to compute 3H and 14C dpm. Findings show that the method presented is adaptable for highly quenched samples up to quenching indices of tSIE = 100. The method is useful for studying the biological transport coupled to Na+.