村田 武士

In F-ATPases, ATP hydrolysis is coupled to translocation of ions through membranes by rotation of a ring of c subunits in the membrane. The ring is attached to a central shaft that penetrates the catalytic domain, which has pseudo-3-fold symmetry. The ion translocation pathway lies between the external circumference of the ring and another hydrophobic protein. The H+ or Na+:ATP ratio depends upon the number of ring protomers, each of which has an essential carboxylate involved directly in ion translocation. This number and the ratio differ according to the source, and 10, 11, and 14 protomers have been found in various enzymes, with corresponding calculated H+ or Na+:ATP ratios of 3.3, 3.7, and 4.7. V-ATPases are related in structure and function to F-ATPases. Oligomers of subunit K from the Na+-motive V-ATPase of Enterococcus hirae also form membrane rings but, as reported here, with 7-fold symmetry. Each protomer has one essential carboxylate. Thus, hydrolysis of one ATP provides energy to extrude 2.3 sodium ions. Symmetry mismatch between the catalytic and membrane domains appears to be an intrinsic feature of both V- and F-ATPases.

V-type Na+-ATPase of Enterococcus hirae binds about six (6 ± 1) Na+ ions/enzyme molecule with a high affinity (Murata, T., Igarashi, K., Kakinuma, Y., and Yamato, I. (2000) J. BioL Chem. 275, 13415-13419). After the addition of 5 mM ATP, the binding capacity dropped to about 2 (1.8 ± 0.3) Na+ ions/enzyme molecule, returning to the initial value concomitant with the decrease of ATP hydrolysis rate. These findings suggest that the affinity of four of six Na+-binding sites of the enzyme changes (lowers) in enzyme reaction. The ATP analogs (adenosine 5′-O-(3-thiotriphosphate) or 5′-adenylylimido-diphosphate), ADP, or aluminum fluoride that is postulated to trap ATPases at their transition state did not inhibit the Na+ binding capacity significantly. Therefore, the affinity decrease of Na+-binding sites was unlikely to be due to ATP binding alone or at the transition state of ATP hydrolysis. In the presence of 5 mM ATP, the ATPase showed strong negative cooperativity (nH = 0.16 ± 0.03) for Na+ stimulation of ATPase activity. The Hill coefficient (nH) increased to 1 in parallel to the decrease of ATP concentration in the reaction mixture. Thus, the ATP-dependent affinity change cooperatively occurs in continuous enzyme reaction.

V-ATPases make up a family of proton pumps distributed widely from bacteria to higher organisms. We found a variant of this family, a Na+-translocating ATPase, in a Gram-positive bacterium, Enterococcus hirae. The Na+-ATPase was encoded by nine ntp genes from F to D in an ntp operon (ntpFIKECGABDHJ): the ntpJ gene encoded a K+ transporter independent of the Na+-ATPase. Expression of this operon, encoding two transport systems for Na+ and K+ ions, was regulated at the transcriptional level by intracellular Na+ as the signal. Structural aspects and catalytic properties of purified Na+-ATPase closely resembled those of other V-type H+-ATPases. Interestingly, the E. hirae enzyme showed a very high affinity for Na+ at catalytic reaction. This property enabled the measurement of ion binding to this ATPase for the first time in the study of V- and F-ATPases. Properties of Na+ binding to V-ATPase were consistent with the model that V-ATPase proteolipids form a rotor ring consisting of hexamers, each having one cation binding site. We propose here a structure model of Na+ binding sites of the enzyme. © 2001 Elsevier Science B.V.

Rotation catalysis theory has been successfully applied to the molecular mechanism of the ATP synthase (F0F1-ATPase) and probably of the vacuolar ATPase. We investigated the ion binding step to Enterococcus hirae Na+- translocating V-ATPase. The kinetics of Na+ binding to purified V-ATPase suggested 6 ± 1 Na+ bound/enzyme molecule, with a single high affinity (K(d(Na+)) = 15 ± 5 μM). The number of cation binding sites is consistent with the model that V-ATPase proteolipids form a rotor ring consisting of hexamers, each having one cation binding site. Release of the bound 22Na+ from purified molecules in a chasing experiment showed two phases: a fast component (about two-thirds of the total amount of bound Na+ k(exchange) &gt 1.7 min-1) and a slow component (about one-third of the total k(exchange) = 0.16 min-1), which changes to the fast component by adding ATP or ATPγS. This suggested that about two-thirds of the Na+ binding sites of the Na+- ATPase are readily accessible from the aqueous phase and that the slow component is important for the transport reaction.

A Na+-translocating ATPase was discovered in a gram-positive bacterium Enterococcus hirae. Our biochemical and molecular biological studies revealed that this Na+-ATPase belongs to the vacuolar-type enzyme. Purified Na+-ATPase consisted of nine subunits: NtpA, B, C, D, E, F, G, I, and K; reconstituted proteoliposomes showed ATP-driven electrogenic Na+ translocation. All these subunits were encoded by the ntp operon: ntpFIKECGABDHJ. The deduced amino acid sequences of the major subunits, A, B, and K (16 kDa proteolipid), were highly similar to those of A, B, and proteolipid subunits of vacuolar ATPases, although the similarities of other subunits were moderate. The ntpJ gene encoded a K+ transporter independent of the Na+-ATPase. Expression of this operon, encoding two transport systems for Na+ and K+ ions, was regulated at transcriptional level by intracellular Na+ as the signal. Two related cation pumps, vacuolar Na+-ATPase and F0F1, H+-ATPase, coexist in this bacterium.

Vacuolar ATPases make up a family of proton pumps distributed widely from bacteria to higher organisms. An unusual member of this family, a sodium-translocating ATPase, has been found in the eubacterium Enterococcus hirae. We report here the purification of enterococcal Na+-ATPase from the plasma membrane of cells, whose ATPase content was highly amplified by expression of the cloned ntp operon that encodes this Na+-ATPase (ntpFIKECGABDHJ). The purified enzyme appears to consist of nine Ntp polypeptides, all the above except for the ntpH and ntpJ gene products. ATPase activity was strictly dependent on the presence of Na+ or Li+ ions and was inhibited by nitrate, N-ethylmaleimide, and the peptide antibiotic destruxin B. When the purified ATPase was reconstituted into liposomes prepared from Enterococcus faecalis phospholipids, ATP-driven Na+ uptake was observed; uptake was blocked by nitrate, destruxin B, and monensin, but it accelerated by carbonyl cyanide m-chlorophenylhydrazone and valinomycin. These data demonstrate that E. hirae Na+-ATPase is an electrogenic sodium pump of the vacuolar type. This is a promising system for research on the fundamental molecular structure and mechanism of vacuolar ATPase.

The ntpJ gene, the tail end in the vacuolar type Na+-ATPase (ntp) operon of Enterococcus hirae, encodes a putative 49-kDa hydrophobic protein resembling K+ transporter protein in Saccharomyces cerevisiae (Takase, K., Kakinuma, S., Yamato, I., Konishi, K., Igarashi, K., and Kakinuma, Y. (1994) J. Biol. Chem. 269, 11037-11044). Northern blotting experiment revealed that the ntpJ gene was transcribed as a cistron in the ntp operon. We constructed an Enterococcus strain in which the ntpJ gene was disrupted by cassette mutagenesis with erythromycin resistance gene. The growth of this mutant was normal at low pH. However, the mutant did not grow at high pH in K+ -limited medium (less than 1 mM), while the wild type strain grew well the internal K+ concentration of this mutant was as low as 7% of that of the wild type strain, suggesting that the K+ accumulation at high pH was inactivated by disruption of the ntpJ gene. Potassium uptake activity via the KtrII system, which had been proposed as the proton potential-independent, Na+-ATPase-coupled system working at high pH (Kakinuma, Y., and Harold, F. M. (1985) J. Biol. Chem. 260, 2086-2091), was missing in this mutant strain. However, this mutant retained as high activities of Na+-ATPase and Na+ pumping as the wild type strain. From these results, we conclude that the NtpJ is a membraneous component of the KtrII K+ uptake system but not a functional subunit of vacuolar Na+-ATPaSe complex the interplay between the KtrII system and the Na+-ATPase was discussed.