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

Abstract

The main electric 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'-disulfonic 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 Km is 1.8 mM and the pH optimum is 6.8, but trypsin treatment of these membranes or their purification on a sucrose gradient decreases both the apparent Km (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 Km is 1.1 mM; at pH 7.8, NO3- increases the Vmax 24-fold. TTP protects against inhibition by DIDS, and the KD for TTP could be estimated to be 0.25 mM, a value close to the apparent Km measured in the same purified membrane preparation. Thiamine pyrophosphate (0.1 mM) did not protect against DIDS inhibition. At lower (10(-5)-10(-6) M) 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 [gamma-32P]TTP. Our data suggest the existence of a high-affinity binding site (KD of approximately 0.5 microM) for the Mg-TTP complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolism of Thiamine Triphosphate in Rat Brain: Correlation with Chloride Permeability

“…Whereas TDP and TMP are also partly bound, thiamine essentially exists in free form. This result is in agreement with our previous studies (Bettendorff et al, 1989), which showed the existence of a specific TTP binding site in Electrophorus electric organ membranes using [y-32P]TTP. The dissociation constant of TTP for its binding site was about 0.5 pM.…”

“…Although the enzymatic hydrolysis of TTP has been studied extensively in rat brain (Barchi and Braun, 1972;Hashitani and Cooper, 1972) and in the electric organ of Etectrophorus dectricus (Bettendorff et al, 1988(Bettendorff et al, , 1989, the mechanism of TTP synthesis remains enigmatic. Early reports (Ruenwongsa and Cooper, 1977) suggesting that TTP could be synthesized in vitro from thiamine diphosphate (TDP) and ATP have not been confirmed (Schrijver et al, 1978; results from our laboratory), partly because quantitative procedures for TTP estimation were unreliable.…”

Thiamine Triphosphate Synthesis in Rat Brain Occurs in Mitochondria and Is Coupled to the Respiratory Chain

“…Membrane-bound ThTPases are present in many animal tissues, including rat brain (15,16), but so far, those enzymes could be neither purified nor characterized at the molecular level. However, a soluble 25-kDa cytosolic ThTPase has been purified from bovine brain (17) and characterized at the molecular level (18).…”

Molecular Characterization of a Specific Thiamine Triphosphatase Widely Expressed in Mammalian Tissues

“…Animal tissues contain a membrane-associated as well as a soluble thiamine triphosphatase (ThTPase; EC 3.6.1.28). The membrane-bound ThTPase (11)(12)(13) has not been purified, and its specificity for ThTP remains uncertain. The soluble ThTPase first described by Hashitani and Cooper (14) in rat brain has been purified to homogeneity from bovine brain (15) and kidney (16).…”