CROSS-LINKING INDUCES HOMODIMER FORMATION AND INHIBITS ENZYMATIC-ACTIVITY OF CHICKEN STOMACH ECTO-APYRASE

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