Single-molecule Study on the Temperature-sensitive Reaction of F1-ATPase with a Hybrid F1 Carrying a Single ��(E190D)

F₁-ATPase is a rotary molecular motor in which the γ-subunit rotates against the α₃β₃ cylinder. The unitary γ-rotation is a 120° step comprising 80 and 40° substeps, each of these initiated by ATP binding and ADP release and by ATP hydrolysis and inorganic phosphate release, respectively. In our previous study on γ-rotation at low temperatures, a highly temperature-sensitive (TS) reaction step of F₁-ATPase from thermophilic Bacillus PS3 was found below 9 °C as an intervening pause before the 80° substep at the same angle for ATP binding and ADP release. However, it remains unclear as to which reaction step the TS reaction corresponds. In this study, we found that the mutant F₁(βE190D) from thermophilic Bacillus PS3 showed a clear pause of the TS reaction below 18 °C. In an attempt to identify the catalytic state of the TS reaction, the rotation of the hybrid F₁, carrying a single copy of βE190D, was observed at 18 °C. The hybrid F₁ showed a pause of the TS reaction at the same angle as for the ATP binding of the incorporated βE190D, although kinetic analysis revealed that the TS reaction is not the ATP binding step. These findings suggest that the TS reaction is a structural rearrangement of β before or after ATP binding.F₁-ATPase (F₁)² is an ATP-driven rotary motor protein. The subunit composition of the bacterial F₁-ATPase is α₃β₃γδϵ, and the minimum complex of F₁-ATPase as a rotary motor is α₃β₃γ subcomplex. This motor protein forms the F_oF₁-ATP synthase complex by binding to another rotary motor, namely, F_o, which is driven by the proton flux resulting from the proton motive force across the membranes (1–4). Under physiological conditions, where the proton motive force is sufficiently large, F_o forcibly rotates F₁-ATPase in the reverse direction of F₁-ATPase, leading the reverse reaction of ATP hydrolysis, i.e. ATP synthesis from ADP and inorganic phosphate (P_i). When the proton motive force diminishes or F₁ is isolated from F_o, F₁-ATPase hydrolyzes ATP to rotate the γ-subunit against the α₃β₃ stator ring in the counterclockwise direction as viewed from the F_o side (5). The catalytic sites are located at the interface of the α- and β-subunits, predominantly on the β-subunit (6). Each β-subunit carries out a single turnover of ATP hydrolysis during the γ-rotation of 360° following the common catalytic reaction pathway, whereas they are 120° different in the catalytic phase. In this manner, the three β-subunits undergo different reaction steps of ATP hydrolysis upon each rotational step. The rotary motion of the γ-subunit has been demonstrated by biochemical (7) and spectroscopic methods (8) and directly proved in single-molecule observation studies (5).Since the establishment of the single-molecule rotation assay, the chemomechanical coupling scheme of F₁ has been studied extensively by resolving the rotation into discrete steps. The stepping rotation was first observed under an ATP-limiting condition where F₁ makes discrete 120° steps upon ATP binding (9). Then, high speed imaging of the rotation with a small probe of low friction was performed, which revealed that the 120° step comprises 80 and 40° substeps, each initiated by ATP binding, and two unknown consecutive reactions, respectively (10). This finding necessitated the identification of the two reactions that trigger the 40° substep. Hence, the rotation assay was performed using a mutant, namely F₁(βE190D), and a slowly hydrolyzed ATP analog, namely ATPγS (11). Glutamate 190 of the β-subunit of F₁, derived from thermophilic Bacillus PS3 and the corresponding glutamates from other F₁-ATPases (Glu-181 of F₁ from Escherichia coli and Glu-188 of F₁ from bovine mitochondria), has been identified as one of the most critical catalytic residues for ATP hydrolysis (6, 12–15). When this glutamate was substituted with aspartic acid, which has a shorter side chain than that of glutamate, the ATP cleavage step of F₁ was drastically slowed. In the rotation assay, this mutant showed a distinct long pause before the 40° substep. ATPγS also caused a long pause before the 40° substep. These observations established that the 40° substep is initiated by hydrolysis. Accordingly, the pause angles before the 80 and 40° substeps are referred to as to the binding angle and the catalytic angle, respectively. Then, the rotation assay was performed in the presence of a high amount of P_i in the solution. It was shown that P_i rebinding caused the long pause at the catalytic angle, suggesting that P_i is released before the 40° substep (16).However, the reaction scheme of F₁ cannot be established by simply assigning each reaction step to either the binding angle or the catalytic angle, because each reaction step must be assigned to one of the three binding or catalytic angles when considering the 360° cyclic reaction scheme of each β-subunit. Direct information about the timing of ADP release was obtained by simultaneous imaging of fluorescently labeled nucleotides and γ rotation, which showed that each β retains ADP until the γ rotates 240° after binding of the nucleotide as ATP and releases ADP between 240 and 320° (16, 17). Another powerful approach is the use of a hybrid F₁ carrying a mutant β that causes a characteristic pause during the rotation. In a previous study, the hybrid F₁ carrying a single copy of β(E190D), α₃β₂β(E190D)γ, showed a distinct pause caused by the slow hydrolysis of β(E190D) at +200° from the ATP binding angle of the mutant β (18). From this observation, it was confirmed that each β executes the chemical cleavage of the bound ATP at +200° from the angle where the ATP binds to β. The asymmetric feature of the pause of the hybrid F₁ was also utilized in other experiments as a marker in the rotational trajectory to correlate the rotational angle and the conformational state of β (19) or to determine the state of F₁ in the crystal structures as the pausing state at catalytic angle (20).Recently, we have found a new reaction intermediate of F₁ rotation as a clear intervening pause before the 80° substep in the rotation assay below 9 °C (21). Furuike et al. (22) also observed the TS reaction in a high speed imaging experiment. The rate constant of this reaction was remarkably sensitive to temperature, giving a Q₁₀ factor around 19. When ADP was added to solution, the pause before the 80° substep was prolonged, whereas the solution P_i caused a longer pause before the 40° substep (21). Although this result can be explained by assuming that the temperature-sensitive (TS) reaction is ADP release, it was not decisive for the identification of the TS reaction.In this study, we found that the mutant F₁(βE190D) also exhibits the distinct pause of the TS reaction but at a higher temperature than for the wild-type F₁, i.e. at 18 °C. This feature was advantageous in identifying the angle position of the TS reaction in the catalytic cycle for each β-subunit coupled with the 360° rotation. Taking advantage of the feature of the hybrid F₁, we analyzed the rotational behavior of the hybrid F₁ at 18 °C in order to assign the angle position of the TS reaction in the catalytic cycle of the 360° rotation, and we have shown that the TS reaction is not directly involved in the ADP release but in some conformational rearrangement before or after ATP binding step.     

Establishment and characterization of cell lines derived from serous adenocarcinoma (JHOS-2) and clear cell adenocarcinoma (JHOC-5, JHOC-6) of human ovary

The cell lines designated JHOS-2, JHOC-5 and JHOC-6 were established from epithelial ovarian carcinomas. JHOS-2 was established from a serous adenocarcinoma of a 45-year-old Japanese woman, JHOC-5 from a recurrent tumor of a clear cell adenocarcinoma of a 47-year-old Japanese woman and JHOC-6 from a tumor of a clear cell adenocarcinoma of a 43-year-old Japanese woman. These cell lines have grown well and serial passages were successively carried out more than 20 times. The monolayer cultured cells revealed neoplastic and pleomorphic features, and grew in multilayers. Electron micrographs revealed epithelial origins that had desmosomes and tonofilaments.     

Inhibition of motility and invasiveness of renal cell carcinoma induced by short interfering RNA transfection of beta 1,4GalNAc transferase

Human renal cell carcinoma (RCC) has been characterized by remarkable changes in ganglioside composition. TOS1 cells, typical of metastatic RCC, are characterized by predominance of GM2 as monosialoganglioside, and beta 1,4GalNAc disialyl-Lc(4) (RM2 antigen) as disialoganglioside [J. Biol. Chem. 276 (2001) 16695]. In order to observe the functional role of gangliosides in RCC malignancy, TOS1 cells were transfected with short interfering RNA (siRNA) based on open reading frame sequence of beta 1,4GalNAc transferase (beta 1,4GalNAc-T), and its disordered sequence of siRNA (dsiRNA) as control. In siRNA transfectant, beta 1,4GalNAc-T mRNA level and GM2 expression were greatly reduced, whereby GM3 expression appeared. In contrast, RM2 antigen level was unchanged, even though it has the same beta 1,4GalNAc epitope at the terminus. dsiRNA transfectant showed no change of beta 1,4GalNAc-T mRNA and did not express GM3. Concomitant with reduction of GM2 and appearance of GM3, siRNA transfectant showed greatly reduced motility and invasiveness, although growth rate was unaltered. Both transfectants with siRNA and dsiRNA expressed the same level of tetraspanin CD9. Since CD9/GM3 complex is known to reduce integrin-dependent motility and invasiveness [Biochemistry 40 (2001) 6414], it is plausible that motility and invasiveness of siRNA transfectant of TOS1 cells may be reduced by enhanced formation of such complex.     

Production of age-related DNA strand breakage in brain cells of senescence-accelerated prone (SAMP1) mouse.

Amounts of DNA strand breaks were estimated by the proportion of cells without tails (PCWT) and the average lengths of tail momentum (ALTM) in comet images of tissue cells of senescence-accelerated prone (SAMP1) mouse and senescence-accelerated resistant (SAMR1) mouse. The PCWT and ALTM of brain cells from SAMR1 were unchanged from 4 to 15 months of age. In the case of SAMP1 brain cells, the PCWT decreased and the ALTM increased in an age-related manner from 8 to 15 months of age. In the cases of liver and kidney, the PCWT and the ALTM of both SAMP1 and SAMR1 cells showed constant values from 4 to 15 months of ages.     

Cyclic AMP metabolism in intact rat ventricular cardiac myocytes: Interaction of carbachol with isoproterenol and 3-isobutyl-1-methylxanthine

Experiments were carried out to elucidate the characteristics of regulation of cyclic AMP levels in intact myocardial cells. For this purpose, the influence of isoproterenol, a nonselective cyclic nucleotide phosphodiesterase (PDE) inhibitor 3-isobutyl-1-methylxanthine (IBMX) and carbachol on cyclic AMP levels was investigated in isolated rat cardiac myocytes. The extent of cyclic AMP accumulation induced by isoproterenol was much less than that produced by IBMX: submaximal concentrations of isoproterenol and IBMX elevated the cyclic AMP level 2.4- and 4.8-fold of the control level, respectively. Both agents in combination increased the cyclic AMP level markedly 48-fold. Carbachol inhibited the cyclic AMP accumulation induced by isoproterenol, IBMX and their combination by 30%, 60% and 80% of the respective response. The extent of inhibition produced by carbachol of the cyclic AMP accumulation induced by IBMX + isoproterenol was smaller than that caused by propranolol, and carbachol produced only a marginal additional inhibitory action to that of propranolol, implying that carbachol does not affect the process of cyclic AMP degradation. The present findings indicate that in intact cardiac myocytes the rate of cyclic AMP degradation catalyzed by PDE may be a crucial process of cyclic AMP turnover. This view is supported by the observations that the inhibitory action of carbachol on the effect of isoproterenol was less than that on the effect of IBMX, and that the inhibitory action of carbachol was markedly enhanced by the simultaneous presence of IBMX.     

Frequency of mutant T lymphocytes defective in the expression of the T-cell antigen receptor gene among radiation-exposed people.

The frequency of mutant T lymphocytes defective in T-cell receptor gene (alpha or beta) expression was measured using the 2-color flow cytometric technique. Results for a total of 203 atomic bomb survivors, 78 of whom were proximally exposed (DS86 doses of greater than or equal to 1.5 Gy) and 125 of whom were distally exposed (DS86 dose of less than 0.005 Gy), showed that the mutant frequency was significantly higher in males than in females. No significant dose effects were observed. In contrast, a significant increase of mutant frequency was observed for 6 patients treated with Thorotrast, a contrast medium containing thorium-232 formerly used for radioligands. In addition, thyroid disease patients treated with 131I showed a dose-related increase of mutant frequency. It was suggested that the present T-cell receptor mutation assay has a unique characteristic as a biological dosimeter for measurement of recent exposures to genotoxic agents.