ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 259, No. 2, December, pp. 650-653, 1987 COMMUNICATION The Halobacterial H+-Translocating ATP Synthase Relates to the Eukaryotic Anion-Sensitive H+-ATPase’ YASUO MUKOHATA; KUNIO IHARA, MASASUKE YOSHIDA,* JIN KONISHI,8, YASUO SUGIYAMA, AND MANABU YOSHIDA Department of Biology, Faculty of Science, Osaka, University, Toyonaka 560 and *Department oj’ Life Science, Tokyo Institute of Technology, Yokohama 229, Japan Received August 21, 198’7 The H+-translocating ATP synthase of Halobacterium halobium (Y. Mukohata and M. Yoshida (1987) J. Biochem. 102,797~802) includes a catalytic moiety of 320 kDa which is isolated as an azide-insensitive ATPase (T. Nanba and Y. Mukohata (1987) J. Biochem. 102,591-598). The polyclonal antibody against this archaebacterial ATPase cross-reacts with the anion-sensitive H+-ATPase of red beet, Beta vzclgaris, tonoplast as well as with another archaebacterial ATPase from Sulfolobus acidocaldarius. The affinity is much higher than to F1-ATPase from spinach chloroplasts or to Ca’+-ATPase from sarco- plasmic reticulum of rabbit skeletal muscle. D 1987 Academic press, I~~. The extremely halophilic archaebacterium Halo- bacterium halo&urn synthesizes ATP by a H+-trans- locating ATP synthase (1) that apparently differs from FOF1-ATPase synthase (2). The ATP synthase is inhibited by dicyclohexylcarbodiimide (DCCD)3 (3,4) but not by azide nor by vanadate (5), the specific inhibitors for FaFi-ATPase synthase and cation- transporting EiEz-ATPases, respectively. DCCD- binding polypeptides of 78 and 12 kDa have been identified (6), one of which seems to be the part of the synthase that anchors the enzyme to the membrane. An ATPase of 320 kDa was isolated from the plasma membrane of H. halo&urn; it is likely com- posed of two pairs of subunits, of 86 and 64 kDa, and is insensitive to azide and vanadate (7). This ATPase was identified as the (catalytic) portion of the ATP i This work was supported in part by a Grant-in- Aid for Scientific Research on Priority Areas of “Bioenergetics”(62617003) from the Ministry of Edu- cation, Science and Culture of Japan. ‘To whom correspondence should be addressed. a Abbreviations used: DCCD, dicyclohexylcarbodi- imide; NBD-Cl, 7-chloro-4-nitrobenzo-2-oxa-1,3-dia- zole; SDS, sodium dodecyl sulfate; CBB, Coomassie brilliant blue; PMF, proton motive force. synthase (2), so halobacterial H+-translocating ATP synthase is the first exception from the common perception that FOF,-ATPase synthase is universal in the energy-transducing membranes of respiring or- ganisms An attempt has been made to understand the (evolutionary) status of this enzyme among other ion-translocating ATPases. The ATPases from other archaebacteria, H. saccharworum (8), Sulfolobus aci- docaldarius (9, lo), and Methanosarcina barkeri (ll), are also insensitive to azide and vanadate. These en- zymes have two major subunits (Table I), are active in acidic pH, and are stimulated by sulfite and inhib- ited by nitrate. 7-Chloro-4-nitrobenzo-2-oxa-1,3-dia- zole (NBD-Cl) specifically inactivates the ATP synthase in situ (5), purified ATPase (7) of halobac- teria, and the ATPase from Sulfoloh~s (10). The inhi- bition of the halobacterial enzymes is partially pre- vented when ATP is added with the modifier (2, 5). The H+-ATPases insensitive to azide and vanadate have also been found in the membranes of storage, secretory, and regulatory organelles such as vacu- oles, Golgi bodies, synaptosomes, and chromaffin granules (12). These ATPases are called anion-sensi- tive H”-ATPases (13) since they are commonly stim- ulated by chloride and inhibited by nitrate. These ATPases also commonly contain (at least) two major 0003-9861/87 $3.00 650 Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.