F0F1-ATPase of Vibrio parahaemolyticus: purification using new detergents and characterization.
Previous attempts to isolate a stable F0F1-ATPase complex (H(+)-translocating ATPase) from Vibrio parahaemolyticus have been unsuccessful. Using new non-ionic detergents (alkyl thiomaltosides), a stable F0F1 complex with a high specific activity (15-25 units/mg protein) was purified and characterized. The purified F0F1-ATPase consists of eight subunits (alpha, beta, gamma, delta, epsilon, a, b and c). The new detergents, in combination with sucrose (or glycerol), lipid, dithiothreitol and phenylmethylsulfonyl fluoride, effectively stabilized the F0F1 complex. The ATPase activity of the F0F1 complex was greatly increased by anions, such as SO4(2-) and SO3(2-). Sodium ion increased the activity by about 2-fold. Dicyclohexylcarbodiimide, Zn2+, 4-acetamido-4'-isothiocyanostilben-2,2'disulfonate and tetrachlorosalicylanilide inhibited F0F1-ATPase activity. Ethanol, which stimulated F1-ATPase activity, inhibited F0F1-ATPase activity. Methanol, Na3VO4 and bafilomycin A1 did not have any significant effect on F0F1-ATPase activity, although methanol, like ethanol, stimulated F1-ATPase activity.
Ogawa
W
W
Department of Microbiology, Faculty of Pharmaceutical Sciences, Okayama University, Japan.
Izawa
S
S
Sakai-Tomita
Y
Y
Moritani
C
C
Tsuda
M
M
Kinomura
K
K
Kitazawa
S
S
Tsuchiya
T
T
D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000494:pharmacology; D003902:Detergents; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D011510:Proteolipids / Q000737:chemistry; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D012995:Solubility; D025262:Vacuolar Proton-Translocating ATPases; D014736:Vibrio parahaemolyticus / Q000201:enzymology*; D015032:Zinc / Q000494:pharmacology
D003902:Detergents; D011510:Proteolipids; C082471:ductin; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D004024:Dicyclohexylcarbodiimide; D025262:Vacuolar Proton-Translocating ATPases; D006180:Proton-Translocating ATPases; D015032:Zinc