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.

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Fish skin mucus contains a variety of antimicrobial proteins and peptides that seem to play a role in self defense. We previously reported an antibacterial protein in the skin secretion of the rockfish, Sebastes schlegeli, which showed selective antibacterial activity against Gram‐negative bacteria. This study aimed to isolate and structurally and functionally characterize this protein. The antibacterial protein, termed SSAP (S. schlegeli antibacterial protein), was purified to homogeneity by lectin affinity column chromatography, anion‐exchange HPLC and hydroxyapatite HPLC. It was found to be a glycoprotein containing N‐linked glycochains and FAD. Its molecular mass was estimated to be 120 kDa by gel filtration HPLC and 53 kDa by SDS/PAGE, suggesting that it is a homodimer. On the basis of the partial amino‐acid sequence determined, a full‐length cDNA of 2037 bp including an ORF of 1662 bp that encodes 554 amino‐acid residues was cloned by 3′ RACE, 5′ RACE and RT‐PCR. A blast search showed that a mature protein (496 residues) is homologous to l‐amino acid oxidase (LAO) family proteins. SSAP was determined to have LAO activity by the H2O2‐generation assay and substrate specificity for only l‐Lys with a Km of 0.19 mm. It showed potent antibacterial activity against fish pathogens such as Aeromonas hydrophila, Aeromonas salmonicida and Photobacterium damselae ssp. piscicida. The antibacterial activity was completely lost on the addition of catalase, confirming that H2O2 is responsible for the growth inhibition. This study identifies SSAP as a new member of the LAO family and reveals LAO involvement in the innate immunity of fish skin.

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The structure of the tryptophan synthase ␣-subunit from Pyrococcus furiosus was determined by x-ray analysis at 2.0-Å resolution, and its stability was examined by differential scanning calorimetry. Although the structure of the tryptophan synthase ␣ 2 ␤ 2 complex from Salmonella typhimurium has been already determined, this is the first report of the structure of the ␣-subunit alone. The ␣-subunit from P. furiosus (Pf-␣-subunit) lacked 12 and 6 residues at the N and C termini, respectively, and one residue each in two loop regions as compared with that from S. typhimurium (St-␣-subunit), resulting in the absence of an N-terminal helix and the shortening of a C-terminal helix. The structure of the Pf-␣-subunit was essentially similar to that of the St-␣-subunit in the ␣ 2 ␤ 2 complex. The differences between both structures were discussed in connection with the higher stability of the Pf-␣-subunit and the complex formation of the ␣-and ␤-subunits. Calorimetric results indicated that the Pf-␣-subunit has extremely high thermostability and that its higher stability is caused by an entropic effect. On the basis of structural information of both proteins, we analyzed the contributions of each stabilization factor and could conclude that hydrophobic interactions in the protein interior do not contribute to the higher stability of the Pf-␣-subunit. Rather, the increase in ion pairs, decrease in cavity volume, and entropic effects due to shortening of the polypeptide chain play important roles in extremely high stability in Pf-␣-subunit.Prokaryotic tryptophan synthase, which catalyzes the last processes in the biosynthesis of tryptophan, is a multienzyme ␣ 2 ␤ 2 complex composed of nonidentical ␣-and ␤-subunits. The separate ␣-and ␤ 2 -subunits catalyze inherent reactions termed ␣ and ␤ reactions, respectively. When the ␣-and ␤ 2 -subunits combine to form the ␣ 2 ␤ 2 complex, the enzymatic activity of each subunit is stimulated by 1 to 2 orders of magnitude (1). The ␣ 2 ␤ 2 complex has been studied as an excellent model system for seeking answers to important questions in proteinprotein interaction, especially in multifunctional enzymes. In 1988 (2) the three-dimensional structure of the tryptophan synthase ␣ 2 ␤ 2 complex from Salmonella typhimurium was determined by x-ray analysis. However, the structure of the ␣-or ␤ 2 -subunit alone has not yet been determined. To elucidate the molecular basis of the mutual activation of the subunit interaction due to the formation of the ␣ 2 ␤ 2 complex, we need to know the structures of the ␣-or ␤ 2 -subunits alone as well as that of the complex. Although the crystallization of each subunit from S. typhimurium and Escherichia coli has been tried for many years (3), the report of the x-ray structure has not yet appeared. Recently, the structures of a number of proteins from hyperthermophiles have been successfully determined by x-ray analysis. This seems due to the facts that proteins from hyperthermophiles are unusually stable and more easily form better crystals. Therefore, the ...