Cross‐linking induces homodimer formation and inhibits enzymatic activity of chicken stomach ecto‐aryrase

We have investigated the effect of cross‐linking on the enzymatic activity and oligomer formation of the chicken stomach ecto‐apyrase. Cross‐linking with the hydrophobic, lysine‐specific dithiobis(succinimidylpropionate) (DSP) caused equal inhibition of ATPase and ADPase activity in both the membrane‐bound and detergent‐solubilized ecto‐apyrase. The inhibitory effect of cross‐linking was reversed upon the addition of the reductant dithiothreitol. Western blots of aliquots of the cross‐linked samples show decreased amounts of the monomeric 80 kDa ecto‐apyrase and the appearance of a 160 kDa dimer under conditions inducing enzyme inhibition. Therefore, the chicken stomach ecto‐apyrase, like the chicken gizzard ecto‐ATPase, is likely a homodimer in vivo. Unlike the related gizzard ecto‐ATPase, however, the native stomach ecto‐apyrase is not stimulated, but rather inhibited by cross‐linking, presumably due to different quaternary structural stability of the two enzymes.