Intramolecular cross-linking at the active site of the Ca2+-ATPase of sarcoplasmic reticulum. High and low affinity nucleotide binding and evidence of active site closure in E2-P.
Limited reaction of glutaraldehyde with the Ca2+-ATPase (Mr approximately 110,000) of sarcoplasmic reticulum results in intramolecular cross-linking at the active site, which can be detected by an anomalous increase in apparent molecular weight (Mr approximately 125,000) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Ross D.C., and McIntosh D.B. (1987) J. Biol. Chem. 262, 2042-2049). ATP, ADP, AMPPCP, trinitrophenyladenosine triphosphate, and decavanadate inhibited the cross-link in a manner suggestive of a homogeneous class of inhibitory sites, with K0.5 values for inhibition in agreement with Kd values for binding to the active site. Cross-link formation was inhibited in proportion to phosphoenzyme levels formed from Pi (E2-P) whereas stoichiometric phosphorylation from CaATP (E1-P) had no effect. Inhibition was observed at millimolar concentrations of CaATP, indicative of nucleotide binding to E1-P. MgATP, in the presence of Ca2+, inhibited cross-linkage in the micromolar and millimolar concentration ranges, the former attributable to E1 X ATP and E2-P formation and the latter to ATP binding mainly to E1-P. The inability to cross-link the active site only of the E2-P intermediate suggests a unique active site conformation, possibly a closed active site cleft, which we suggest is linked to low affinity, inwardly orientated Ca2+-binding sites.
D000244:Adenosine Diphosphate / Q000494:pharmacology*; D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000447:Aldehydes / Q000494:pharmacology*; D000818:Animals; D001665:Binding Sites; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D003432:Cross-Linking Reagents; D005976:Glutaral / Q000494:pharmacology*; D007700:Kinetics; D009132:Muscles / Q000201:enzymology; D010710:Phosphates / Q000494:pharmacology; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D014638:Vanadates / Q000494:pharmacology
D000447:Aldehydes; D003432:Cross-Linking Reagents; D010710:Phosphates; D014638:Vanadates; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D005976:Glutaral