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Persistent Ca2(+)-induced activation of erythrocyte membrane Ca2(+)-ATPase unrelated to calpain proteolysis.
- Source :
-
Archives of biochemistry and biophysics [Arch Biochem Biophys] 1990 May 15; Vol. 279 (1), pp. 78-86. - Publication Year :
- 1990
-
Abstract
- Preincubation of human erythrocyte membranes with calcium in the submillimolar to millimolar concentration range resulted in an increase of the Ca2+ affinity and apparent maximum velocity of the Ca2(+)-stimulated Mg2(+)-dependent ATPase (Ca2(+)-ATPase). The activation was persistent, as it was not reversed when the Ca2(+)-preincubated membranes were washed with ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid-containing buffers. Magnesium was not required for the activation, whereas greater than 2 mM Mg2+ partially antagonized the activation by Ca2+. In some membrane preparations ATP was required in addition to Ca2+ for activation of the Ca2(+)-ATPase, but nonhydrolyzable analogs of ATP had the same effect. Calmodulin prevented the activation by Ca2+ over the same concentration range in which it interacts with the Ca2(+)-ATPase. Taken together the results obtained provided strong evidence that the Ca2+ activation of the enzyme was not due to proteolytic cleavage by endogenous calpain. Thus, activation by Ca2+ was not blocked by leupeptin (100-200 microM), did not require dithiothreitol, and occurred at Ca2+ concentrations greater than those required for activation of calpain I. Furthermore, Ca2+ activation did not result in change in the mobility the native 136-kDa species of the Ca2(+)-ATPase on SDS-gel electrophoresis. Moreover, solubilization of the Ca2(+)-pretreated membranes with Triton X-100 reversed the Ca2+ activation of the Ca2(+)-ATPase. On the other hand, Ca2(+)-pretreatment of the membranes modified the susceptibility of the Ca2(+)-ATPase to both cleavage and activation by exogenously added calpain I. We conclude that pretreatment of Ca2(+)-ATPase in erythrocyte membranes with millimolar Ca2+ activates the enzyme by inducing a persistent conformational change of the enzyme which is, however, subsequently reversed by detergent solubilization.
- Subjects :
- Adenosine Triphosphate analogs & derivatives
Adenosine Triphosphate pharmacology
Enzyme Activation drug effects
Erythrocyte Membrane drug effects
Humans
Models, Biological
Octoxynol
Phosphorylation
Polyethylene Glycols pharmacology
Protein Denaturation
Solubility
Calcium pharmacology
Calcium-Transporting ATPases metabolism
Calpain metabolism
Erythrocyte Membrane enzymology
Subjects
Details
- Language :
- English
- ISSN :
- 0003-9861
- Volume :
- 279
- Issue :
- 1
- Database :
- MEDLINE
- Journal :
- Archives of biochemistry and biophysics
- Publication Type :
- Academic Journal
- Accession number :
- 2140035
- Full Text :
- https://doi.org/10.1016/0003-9861(90)90465-b