Open AccessDemonstration of a Stable Occluded Form of Ca 2+ by the Use of the Chromium Complex of ATP in the Ca 2+ ‐ATPase of Sarcoplasmic Reticulum
Author(s) -
SERPERSU Engin Halit,
KIRCH Ulrïke,
SCHONER Wilhelm
Publication year - 1982
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1982.tb05887.x
Subject(s) - endoplasmic reticulum , chemistry , vesicle , atpase , calcium , ionophore , dissociation constant , enzyme , calcium atpase , dissociation (chemistry) , biochemistry , biophysics , membrane , biology , receptor , organic chemistry
Sarcoplasmic reticulum Ca 2+ ‐ATPase is slowly inactivated by the β, γ‐bidentate of the chromium(III) complex of ATP (CrATP). The rate of inactivation depends on the presence of calcium ions in the incubation medium. Half‐maximal inactivation rate is reached at 80 nm Ca 2+ . The dissociation constant of enzyme‐CrATP complex at 26°C is 31 μM. Inactivation of the enzyme by CrATP is protected competitively by ATP. From this a dissociation constant of the enzyme ATP complex of 6.2 μM is calculated. The inactivation of Ca 2+ ‐ATPase is proportional to the incorporation of 32 P from [γ‐ 32 P]CrATP into the 1‐M r protein. Concomitant with the inactivation there is also an incorporation of 45 Ca 2+ into the purified Ca 2+ ‐ATPase. A ratio of 45 Ca 2+ binding to phosphorylation of 1.7‐1.9 is calculated from the capacities of both processes as well as from their kinetics. When Ca 2+ uptake into sarcoplasmic reticulum is fuelled by ATP, treatment of the vesicles with the Ca 2+ ionophore X‐537‐A or the production of leaks by adding deoxycholate leads to a loss of already accumulated 45 Ca 2+ . Such treatment, however, leaves the 45 Ca 2+ in the vesicles of Ca 2+ ‐ATPase unaffected, if ATP had been replaced by CrATP during the Ca2+ uptake. It is possible to localize 45 Ca 2+ together with the phosphointermediate in the dodecylsulfate electrophoresis on the 1‐M r protein and to a minor degree on the 2‐M r dimer. This apparently occluded 45 Ca 2+ is rapidly lost by treatment of the enzyme with trichloroacetic acid. It is concluded, that CrATP acts as an ATP analogue on Ca 2+ ‐ATPase of sarcoplasmic reticulum. Hydrolysis of CrATP, as in (Na + + K + )‐ATPase [Pauls, H., Bresenbröcker, B., and Schoner, W. (1980) Eur. J. Biochem. 109, 523‐533] leads to a stable phosphointermediate, which appears to bind 45 Ca 2+ in a stable occluded form. Both processes apparently lead to the suicide of the Ca 2+ ‐ATPase.