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The V-type Na+-ATPase of the thermophilic, anaerobic bacterium Caloramator fervidus was purified to homogeneity. The subunit compositions of the catalytic V-1 and membrane-embedded V-0 parts were determined and the structure of the enzyme complex was studied by electron microscopy. The V-1 headpiece consists of seven subunits present in one to three copies, and the V-0 part of two subunits in a ratio of 5:2. An analysis of over 7500 single particle images obtained by electron microscopy of the purified V1V0 enzyme complex revealed that the stalk region, connecting the V-1 and V-0 parts, contains two peripheral stalks in addition to a central stalk. One of the two is connected to the V-0 part, while the other is connected to the first via a bar-like structure that is positioned just above V-0, parallel with the plane of the membrane. Ln projection, this bar seems to contact the central stalk. The data show that the stator structure that prevents rotation of the static part of V-0 relative to V-1 in the rotary catalysis mechanism of energy coupling in ATPases/ATPsynthases is more complex than previously thought. (C) 2000 Academic Press.

A purified protein fraction of Bacillus thuringiensis subsp. aizawai (HD-133)-activated toxin was subjected to amino acid modification by phenylglyoxal as an arginine-directed reagent. The effect of phenylglyoxylation was assayed on the K+-dependent accumulation of leucine into brush border membrane vesicles from Bombyx mori larval midgut. Phenylglyoxal inactivates the toxin in a dose- and time-dependent manner. Loss of inhibitory effect of the toxin followed a pseudo-first-order kinetics. Observed rates of inactivation were linearly dependent upon the square of phenylglyoxal concentration and a second-order constant of 4.17 M−2 min−1 was calculated. These results indicate the involvement of highly reactive arginine residues in the formation of specific toxin domains implicated in the interaction with membrane receptor(s).