apyrase and 3-3--dithiobis(sulfosuccinimidyl-propionate)

Apyrase activity is present in the saliva of haematophagous arthropods. It is related to blood-feeding because of the apyrase ability to hydrolyse ADP, a key component of platelet aggregation. Five apyrases with apparent molecular masses of 88, 82, 79, 68 and 67 kDa were identified in the saliva of the vector of Chagas disease, Triatoma infestans. The large size observed during purification of these enzymes suggested oligomerization. In the present study, we confirmed, using gel-filtration and analytical ultracentrifugation, the presence of apyrase oligomers with molecular masses of 200 kDa in the saliva. Electrophoretic analyses showed that disulphide bonds were involved in homo-oligomerization. In addition, heterogeneity in disulphide bonds and in pI was detected, with the pI ranging from 4.9 to 5.4. The present study gives the first insights into the quaternary structure of soluble apyrases.

Topics: Animals; Apyrase; Chromatography, Gel; Cross-Linking Reagents; Disulfides; Dithiothreitol; Electrophoresis, Polyacrylamide Gel; Protein Structure, Quaternary; Saliva; Succinimides; Triatoma; Ultracentrifugation

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.

Topics: Animals; Antibodies, Monoclonal; Apyrase; Blotting, Western; Cell Membrane; Chickens; Cross Reactions; Cross-Linking Reagents; Digitonin; Dimerization; Dithiothreitol; Immunoblotting; Stomach; Succinimides