Thiamine triphosphatase in the membranes of the main electric organ of Electrophorus electricus: substrate-enzyme interactions.
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)
Bettendorff
L
L
Laboratory of General and Comparative Biochemistry, University of Liège, Belgium.
Grandfils
C
C
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; D000838:Anions; D002458:Cell Fractionation; D002462:Cell Membrane / Q000201:enzymology; D004557:Electric Organ / Q000201:enzymology* / Q000648:ultrastructure; D004593:Electrophorus / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D013837:Thiamin-Triphosphatase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013838:Thiamine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D014357:Trypsin
D000838:Anions; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D013838:Thiamine Triphosphate; D010744:Phosphoric Monoester Hydrolases; D014357:Trypsin; D013837:Thiamin-Triphosphatase; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid