Thiamine Triphosphatase in the Membranes of the Main Electric Organ of Electrophorus electricus: Substrate-Enzyme Interactions

The main eJectric organ of Electrophorus electricus is particularly rich in thiamine triphosphate (TTP). Membrane fractions prepared from this tissue contain a thiamine triphosphatase that is strongly activated by anions and irreversibly inhibited by 4,4′-diisothiocyanostilbene-2.2′-disul-fonic acid (DIDS), an anion transport inhibitor. Kinetic parameters of the enzyme are markedly affected by the conditions of enzyme preparation: In crude membranes, the apparent K_m is 1.8 mM and the pH optimium is 6.8, but trypsin treatment of these membranes or their purification on a sucrose gradient decreases both the apparent A_m (to 0.2 mM) and the pH optimum (to 5.0). Anions such as NO₃ (250 mM) have the opposite effect, i.e., even in purified membranes, the pH optimum is now 7.8 and the K_m is 1.1 mM; at pH 7.8, NO₃ increases the V_max 24-fold. TTP protects against inhibition by DIDS, and the K_D for TTP could be estimated to be 0.25 mM, a value close to the apparent K_m measured in the same purified membrane preparation. Thiamine pyrophosphate (0.1 mM) did not protect against DIDS inhibition. At lower (10^-5-10^-6M) substrate concentrations. Lineweaver-Burk plots of thiamine triphosphatase activity markedly deviate from linearity, with the curve being concave downward. This suggests either anticooperative binding or the existence of binding sites with different affinities for TTP. The latter possibility is supported by binding data obtained using [γ-³²P]TTP. Our data suggest the existence of a high-affinity binding site (K_D of ˜0.5 μM) for the Mg-TTP complex. The membrane-bound thiamine triphosphatase is insensitive to low concentrations of vanadate and is not activated by protonophores. These and other features suggest that this enzyme is quite distinct from most of the known membrane ATPases and that it may have a specific function in excitable cell membranes, possibly in relation to their permeability to certain anions.