A Membrane-associated Thiamine Triphosphatase from Rat Brain

A membrane-associated thiamine triphosphatase in subcellular fractions of the rat brain is described. This enzyme is shown to be different from a less active soluble thiamine triphosphatase previously reported. It is also shown to be distinct from ATPases and from a general nucleoside triphosphatase in the tissues examined. The membraneassociated thiamine triphosphatase activity has an absolute divalent cation requirement which is satisfied by Mg2+, Ca2+, or Mnz+. Maximal activation occurs at a cation to substrate ratio of 1:l. At its optimal pH of 6.5, the enzyme exhibits a K, of 1.0 to 2.0 mM and a Vmax of 4.0 to 8.0 pmoles per mg of protein per hour. The enzyme is heat-labile and is inactivated by low concentrations of sodium deoxycholate. ADP (1OV to lop3 M) strongly inhibits the enzyme. A specific inhibitor of the enzyme, the /3-y-methylene phosphonate analog of thiamine triphosphate, has been synthesized, and its inhibitory characteristics are described.