Open AccessDemonstration of Two Different Reactive Sulfhydryl Groups in the ATP‐Binding Sites of Ca 2+ ‐ATPase of Sarcoplasmic Reticulum by Disulfides of Thioinosine Triphosphates
Author(s) -
PATZELTWENCZLER Rosemarie,
KREICKMANN Harald,
SCHONER Wilhelm
Publication year - 1980
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1980.tb04781.x
Subject(s) - egta , dissociation constant , dithiothreitol , endoplasmic reticulum , chemistry , atpase , binding site , stereochemistry , enzyme , nucleotide , affinity label , calmodulin , calcium atpase , biophysics , biochemistry , crystallography , calcium , biology , receptor , organic chemistry , gene
1. The disulfide of thioinosine triphosphate, (Sno PPP ) 2 , is a substrate of the Ca 2+ ‐pump and the Ca 2+ ‐ATPase of sarcoplasmic reticulum ( K m = 400 μM). 2. Inactivation of Ca 2+ ‐ATPase by the β,γ‐methylene diphosphonate analogue of the disulfide of thioinosine triphosphate, (Sno PP [CH 2 ] P ) 2 , in the presence of (Ca 2+ + Mg 2+ + K + ) is preceeded by a dissociable enzyme inhibitor complex with a dissociation constant of 130 μM for a low‐affinity binding site. ATP protected Ca 2+ ‐ATPase against the inactivation under these conditions with a dissociation constant of 140 μM. 3. Kinetic analysis of the inactivations of Ca 2+ ‐ATPase by (Sno PP [CH 2 ] P ) 2 in the absence of Ca 2+ and Mg 2+ but the presence of K + and EGTA led to the appearance of two nucleotide binding sites with two different inactivation velocities. Inactivation rate constants k 2 were found for the rapid inactivating part ( k′ 2 = 1.44 × 10 −2 s −1 ) and the slow inactivating part ( k” 2 = 1.15 × 10 −3 s −1 ). From the protective effect of ATP under these conditions a high‐affinity ( K d = 48.78 μM) and a low‐affinity ATP binding site ( K d =114 μM) were apparent. 4. The affinity of the analogues to the enzyme is decreased in the sequence: (Sno PPP ) 2 > (Sno PP [NH] P ) 2 > (Sno PP [CH 2 ]P) 2 > (Sno P ) 2 . 5. (Sno PPP ) 2 ‐inactivated Ca 2+ ‐ATPase was reactivated by incubation with dithiothreitol. 6. Inactivation of Ca 2+ ‐ATPase by [γ‐ 32 P](Sno PPP ) 2 in the presence of (Mg 2+ + K + + Ca 2+ ) or (EGTA + K + ) was accompanied by the incorporation of hydroxylamine‐insensitive radioactivity into the acid‐precipitable protein. The enzyme‐bound [γ‐ 32 P]Sno PPP was cleaved by dithiothreitol. 7. It is concluded that (Sno PPP ) 2 and its non‐hydrolyzable analogues (Sno PP [NH] P ) 2 and (Sno PP [CH 2 ] P ) 2 act as ATP affinity labels and form mixed disulfides with a sulfhydryl group within the active site.