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The importance of carboxyl groups on the lumenal side of the membrane for the function of the Ca2+-ATPase of sarcoplasmic reticulum

The importance of carboxyl groups on the lumenal side of the membrane for the function of the Ca2+-ATPase of sarcoplasmic reticulum
The importance of carboxyl groups on the lumenal side of the membrane for the function of the Ca2+-ATPase of sarcoplasmic reticulum
The conventional model for transport of Ca2+ by the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum (SR) involves a pair of binding sites for Ca2+ that change upon phosphorylation of the ATPase from being high affinity and exposed to the cytoplasm to being low affinity and exposed to the lumen. However, a number of recent experiments suggest that in fact transport involves two separate pairs of binding sites for Ca2+, one pair exposed to the cytoplasmic side and the other pair exposed to the lumenal side. Here we show that the carbodiimide 1-ethyl-3-[3-(dimethylamino)-propyl] carbodiimide (EDC) is membrane-impermeable, and we use EDC to distinguish between cytoplasmic and lumenal sites of reaction. Modification of the Ca2+-ATPase in sealed SR vesicles with EDC leads to loss of ATPase activity without modification of the pair of high affinity Ca2+-binding sites. Modification of the purified ATPase in unsealed membrane fragments was faster than modification in SR vesicles, suggesting the presence of more quickly reacting lumenal sites. This was confirmed in experiments measuring EDC modification of the ATPase reconstituted randomly into sealed lipid vesicles. Modification of sites on the lumenal face of the ATPase led to loss of the Ca2+-induced increase in phosphorylation by Pi. It is concluded that carboxyl groups on the lumenal side of the ATPase are involved in Ca2+ binding to the lumenal side of the ATPase and that modification of these sites leads to loss of ATPase activity. The presence of MgATP or MgADP leads to faster inhibition of the ATPase by EDC in unsealed membrane fragments than in sealed vesicles, suggesting that binding of MgATP or MgADP to the ATPase leads to a conformational change on the lumenal side of the membrane.
0021-9258
977-982
Webb, R.J.
84c5ddfd-7185-4dc6-97c8-d37f705bdb95
Khan, Y.M.
1d55a382-a3b5-48ac-bc4c-4e84499217b2
East, J.M.
9fe7f794-1d89-4935-9a99-b831d786056e
Lee, A.G.
0891914c-e0e2-4ee1-b43e-1b70eb072d8e
Webb, R.J.
84c5ddfd-7185-4dc6-97c8-d37f705bdb95
Khan, Y.M.
1d55a382-a3b5-48ac-bc4c-4e84499217b2
East, J.M.
9fe7f794-1d89-4935-9a99-b831d786056e
Lee, A.G.
0891914c-e0e2-4ee1-b43e-1b70eb072d8e

Webb, R.J., Khan, Y.M., East, J.M. and Lee, A.G. (2000) The importance of carboxyl groups on the lumenal side of the membrane for the function of the Ca2+-ATPase of sarcoplasmic reticulum. The Journal of Biological Chemistry, 275 (2), 977-982.

Record type: Article

Abstract

The conventional model for transport of Ca2+ by the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum (SR) involves a pair of binding sites for Ca2+ that change upon phosphorylation of the ATPase from being high affinity and exposed to the cytoplasm to being low affinity and exposed to the lumen. However, a number of recent experiments suggest that in fact transport involves two separate pairs of binding sites for Ca2+, one pair exposed to the cytoplasmic side and the other pair exposed to the lumenal side. Here we show that the carbodiimide 1-ethyl-3-[3-(dimethylamino)-propyl] carbodiimide (EDC) is membrane-impermeable, and we use EDC to distinguish between cytoplasmic and lumenal sites of reaction. Modification of the Ca2+-ATPase in sealed SR vesicles with EDC leads to loss of ATPase activity without modification of the pair of high affinity Ca2+-binding sites. Modification of the purified ATPase in unsealed membrane fragments was faster than modification in SR vesicles, suggesting the presence of more quickly reacting lumenal sites. This was confirmed in experiments measuring EDC modification of the ATPase reconstituted randomly into sealed lipid vesicles. Modification of sites on the lumenal face of the ATPase led to loss of the Ca2+-induced increase in phosphorylation by Pi. It is concluded that carboxyl groups on the lumenal side of the ATPase are involved in Ca2+ binding to the lumenal side of the ATPase and that modification of these sites leads to loss of ATPase activity. The presence of MgATP or MgADP leads to faster inhibition of the ATPase by EDC in unsealed membrane fragments than in sealed vesicles, suggesting that binding of MgATP or MgADP to the ATPase leads to a conformational change on the lumenal side of the membrane.

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Published date: 1 January 2000

Identifiers

Local EPrints ID: 56796
URI: https://eprints.soton.ac.uk/id/eprint/56796
ISSN: 0021-9258
PURE UUID: 873a5ede-02ea-4457-94df-c6b743147b91

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Date deposited: 07 Aug 2008
Last modified: 17 Jul 2017 14:30

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Contributors

Author: R.J. Webb
Author: Y.M. Khan
Author: J.M. East
Author: A.G. Lee

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