The ATP hydrolysis of the V 1 -ATPase of Thermus thermophilus have been investigated with an ATP-regenerating system at 25°C. The ratio of ATPase activity to ATP concentration ranged from 40 to 4000 M; from this, an apparent K m of 240 ؎ 24 M and a V max of 5.2 ؎ 0.5 units/mg were deduced. An apparent negative cooperativity, which is frequently observed in case of F 1 -ATPases, was not observed for the V 1 -ATPase. Interestingly, the rate of hydrolysis decayed rapidly during ATP hydrolysis, and the ATP hydrolysis finally stopped. Furthermore, the inactivation of the V 1 -ATPase was attained by a prior incubation with ADP-Mg. The inactivated V 1 -ATPase contained 1.5 mol of ADP/mol of enzyme.Difference absorption spectra generated from addition of ATP-Mg to the isolated subunits revealed that the A subunit can bind ATP-Mg, whereas the B subunit can (3), clathrin-coated vesicles (4), chromaffine granules (5), and the central vacuoles of yeast (6). They are responsible for vacuolar acidification, which plays an important role in a number of cellular processes (1). V o V 1 -ATPases are also found in the plasma membranes of most archea (7-9) and some kinds of eubacteria (10 -12). Several studies indicate that the physiological role of V o V 1 -ATPases of some archea and the thermophilic eubacterium Thermus thermophilus is ATP synthesis coupled to proton flux across the plasma membranes (7,9,(13)(14)(15). Precise understanding of V o V 1 -ATPases would allow the comparison to F o F 1 -ATPases and the elucidation of the common essential mechanism for the coupling of proton translocation across a membrane with ATP formation. However, several problems, such as the difficulty of obtaining a large amount of pure enzyme from vacuolar membranes and an unstable V 1 moiety (17), have limited our investigation of enzymatic properties of V o V 1 -ATPases.T. thermophilus, originally isolated from a hot spring in Japan, is thermophilic, obligatory aerobic, Gram-negative, and chemoheterotrophic eubacterium (25). Its respiratory chain may include energy coupling Site I (26). This bacterium has a large amount of the V o V 1 -ATPase on the plasma membrane, instead of F o F 1 -ATPase (15).In contrast to eukaryotic equivalents, the V 1 moiety of T. thermophilus is easily detached from the membranes using chloroform treatment and ATPase-active stable complex can be obtained in large amounts (10). Throughout this manuscript, the V 1 moiety from T. thermophilus is refereed to V 1 -ATPase.

The age-adjusted annual incidence of nontraumatic ONFH in Aichi Prefecture was estimated at 138.5 patients.Thus the annual incidence rate in Japan (population 128 million) was 1.91/100,000.

A protein fold, six parallel i~ strands surrounding the central a helix, is likely to be a common structure in protein families known to have a typical set of nucleotide binding consensus sequence motifs A and B and to catalyze ATP-triggered reactions. According to this ATP-triggered protein fold, the conserved Glu (or Asp), which acts as a general base to activate a water molecule for an in-line attack of the -/-phosphate, is at the exit of the second/3 strand. The fifth i~ strand may be involved in propagation of conformational change triggered by ATP hydrolysis.[5]. Indeed, the recently unraveled crystal structure of mitochondrial Fi-ATPase (MF0 [6] clearly showed that the folding topology of the ATP binding domain of MFI-fl subunit is almost the same as that of the recA protein, and that the essential Glu (Glu j88) of MF~-]3 subunit above described is really located at the right position to act as a general base. Here, we propose that proteins containing a typical set of Walker's motifs A and B have the conserved Glu (or Asp) between the two motifs and that their topologies of the ATP binding domains are probably all common.

ATP hydrolyzing activity of a mutant ␣ 3 ␤ 3 ␥ subcomplex of F 0 F 1 -ATP synthase (⌬NC) from the thermophilic Bacillus PS3, which lacked noncatalytic nucleotide binding sites, was inactivated completely soon after starting the reaction (Matsui, T., Muneyuki, E., Honda, M., Allison, W. S., Dou, C., and Yoshida, M. (1997) J. Biol. Chem. 272, 8215-8221). This inactivation is caused by rapid accumulation of the "MgADP inhibited form" which, in the case of wild-type enzyme, would be relieved by ATP binding to noncatalytic sites. We reconstituted F 0 F 1 -ATP synthase into liposomes together with bacteriorhodopsin and measured illumination-driven ATP synthesis. Remarkably, ⌬NC F 0 F 1 -ATP synthase catalyzed continuous turnover of ATP synthesis while it could not promote ATP-driven proton translocation. ATP synthesis by ⌬NC F 0 F 1 -ATP synthase, as well as wild-type enzyme, proceeded even in the presence of azide, an inhibitor of ATP hydrolysis that stabilizes the MgADP inhibited form. The time course of ATP synthesis by ⌬NC F 0 F 1 -ATP synthase was linear, and gradual acceleration to the maximal rate, which was observed for the wild-type enzyme, was not seen. Thus, ATP synthesis can proceed without nucleotide binding to noncatalytic sites even though the rate is sub-maximal. These results indicate that the MgADP inhibited form is not produced in ATP synthesis reaction, and in this regard, ATP synthesis may not be a simple reversal of ATP hydrolysis.The F 0 F 1 -ATP synthase is a ubiquitous enzyme in plasma membranes of bacteria, inner membranes of mitochondria, and thylakoid membranes of chloroplasts which utilizes a transmembrane electrochemical potential difference of protons (⌬H ϩ ) 1 for ATP synthesis (1, 2). It can be reversibly separated into a hydrophilic, water-soluble F 1 part and a hydrophobic, membrane-embedded F 0 part. F 0 conducts protons across the membrane. F 1 , also called F 1 -ATPase, has a subunit composition of ␣ 3 ␤ 3 ␥␦⑀ and shows strong activity of ATP hydrolysis. According to the crystal structure of the major part of beef heart mitochondrial F 1 -ATPase (3), ␣ and ␤ subunits are alternatively arranged to form a hexagonal cylinder with a central cavity through which coiled-coil ␣ helices of the ␥ subunit penetrate. F 1 -ATPase was recently proven to be a "rotary motor enzyme", the first ever found in the biological world; the ␥ subunit rotates within an ␣ 3 ␤ 3 hexagon during ATP hydrolysis (4 -6).The F 1 -ATPase has six nucleotide binding sites. Three of them are catalytic and located mainly on the ␤ subunits. The other three, called noncatalytic sites, are mainly located on the ␣ subunits. The function of the noncatalytic site had been obscure but recent studies indicated its regulatory role as follows. During multiple turnover of ATP hydrolysis, MgADP is prone to be entrapped in a catalytic site producing the MgADP inhibited form of the enzyme, which is inactive in ATP hydrolysis. ATP binding to the noncatalytic sites causes release of this inhibitory MgADP from the affected catalytic s...

Combining mutation and chemical modification, we have introduced Asp, Gln, Cys, S-carboxymethylcysteine (Cax) and S-carbamoylmethylcysteine (Cam) into the uositions of Gl@" and Glt?" of the B subunit of F,-ATPase from the thermouhilic Bucilhu PS3. The steady-state ATPase activities of a& complexes containing these changed/3 subunits were 12% (ElgOCax), 7% (ElgOD), 3% (E19OCam),