Solubilization of thiamine triphosphatase from the electric organ of Electrophorus electricus.
The membrane-associated, anion-regulated thiamine triphosphatase from Electrophorus electricus electric organ can be solubilized by various neutral detergents. Polyoxyethylene ethers are the most effective. Anionic detergents readily inactivate the enzyme. A 6.4-fold increase in specific activity is obtained by successive treatment of crude membranes with octanoyl-N-methylglucamide, which solubilized other proteins, and Lubrol-PX with releases 60% of the thiamine triphosphatase (TTPase) activity. Solubilization by Lubrol-PX rapidly modifies kinetic parameters. The Km, Vmax and pH optimum are decreased. However, the solubilized TTPase may be kept at 0 degrees C for many hours without further change in specific activity. At 35 degrees C, the half-life is still 83 min at pH 5.0, but denaturation becomes rapid at pH greater than or equal to 7. Solubilization modifies anion effects on TTPase activity. The activating effect of nitrate is nearly lost, while inhibition by sulfate is no longer time-dependent.
Bettendorff
L
L
Laboratory of general and comparative Biochemistry, University of Liège, Belgium.
Longrée
I
I
Wins
P
P
Schoffeniels
E
E
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D002413:Cations, Divalent / Q000494:pharmacology; D003902:Detergents / Q000737:chemistry; D004557:Electric Organ / Q000201:enzymology*; D004593:Electrophorus / Q000378:metabolism*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism; D009566:Nitrates / Q000494:pharmacology; D011489:Protein Denaturation; D012995:Solubility; D013431:Sulfates / Q000494:pharmacology; D013837:Thiamin-Triphosphatase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
D002413:Cations, Divalent; D003902:Detergents; D008565:Membrane Proteins; D009566:Nitrates; D013431:Sulfates; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D013837:Thiamin-Triphosphatase; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid