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 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.
Lewis Carl
S A
SA
Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Ohio 45267-0575, USA.
Smith
T M
TM
Kirley
T L
TL
D000818:Animals; D000911:Antibodies, Monoclonal; D001081:Apyrase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000502:physiology*; D015153:Blotting, Western; D002462:Cell Membrane / Q000737:chemistry / Q000201:enzymology; D002645:Chickens; D003429:Cross Reactions; D003432:Cross-Linking Reagents / Q000737:chemistry / Q000494:pharmacology*; D004072:Digitonin / Q000737:chemistry; D019281:Dimerization; D004229:Dithiothreitol / Q000494:pharmacology; D015151:Immunoblotting; D013270:Stomach / Q000201:enzymology*; D013388:Succinimides / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D003432:Cross-Linking Reagents; D013388:Succinimides; C035759:3,3'-dithiobis(sulfosuccinimidyl propionate); D001081:Apyrase; C011240:dithiobis(succinimidylpropionate); D004072:Digitonin; D004229:Dithiothreitol