Calf spleen NAD glycohydrolase. Comparison of the catalytic properties of the membrane-bound and the hydrosoluble forms of the enzyme.

The catalytic properties of membrane-bound calf spleen NAD glycohydrolase were studied in comparison with previous data obtained with a solubilized hydrosoluble form of the enzyme. When the hydrolysis of NAD catalyzed by membrane-bound NAD glycohydrolase was studied at pH values below 7.5, only insignificant interference by other NAD-hydrolyzing enzymes was detected, and no proton-diffusional inhibition was observed. The kinetics could, therefore, be followed using a titrimetric assay for NAD glycohydrolase activity. The effect of pH, ionic strength on the kinetic parameters, and shifts in binding constants for several ligands of the membrane-bound enzyme indicate that the NAD glycohydrolase activity is influenced by an electrostatic potential due to negative charges (polyelectrolyte effect). No significant changes in kinetic mechanism could be found between both NAD glycohydrolase forms. The association of the enzyme with the membrane results in a remarkably increased thermal stability, in changes in binding properties of the active site and in the emergence of new inhibitor binding sites; e.g. adenosine 3':5'-monophosphate (cyclic AMP) and adenosine, which do not inhibit the hydrosoluble form of NAD glycohydrolase, are good inhibitors (respectively competitive and mixed) of the membrane-bound enzyme. These data (i.e. allotopic changes) probably can be ascribed to enzyme conformational changes induced and stabilized by interaction with membrane constituents.