TY - JOUR
T1 - Heterogeneous halothane binding in the SR Ca2+-ATPase
AU - Kosk-Kosicka, Danuta
AU - Fomitcheva, Ioulia
AU - Lopez, Maria M.
AU - Eckenhoff, Roderic G.
N1 - Funding Information:
We thank Drs. Peter Gillespie and Peter Maloney from the Johns Hopkins University for protein scanning. Supported by grant GM 447130 (D.K.K.) and GM 51595 (R.E.) from the National Institutes of Health.
PY - 1997/1/27
Y1 - 1997/1/27
N2 - The activity of various Ca2+-ATPases is affected by volatile anesthetics, such as halothane, commonly used in clinical practice. The effect on the enzyme in skeletal muscle sarcoplasmic reticulum (SR) is biphasic, including stimulation at clinical anesthetic concentrations and subsequent inhibition at higher concentrations. We have previously proposed that the action of a volatile anesthetic on Ca2+-ATPases results from its binding in the interior of the enzyme molecule [Lopez, M.M. and Kosk-Kosicka, D. (1995) J. Biol. Chem. 270, 28239-28245]. Presently, we investigated whether the anesthetic interacts directly with the skeletal muscle SR Ca2+-ATPase (SERCA1) as evidenced by binding. Photoaffinity labeling with [14C]halothane demonstrated that the anesthetic binds saturably to SR membranes, and that ~80% of the binding is specific, with a K(I) of 0.6 mM. The K(I) value agrees well with the concentration at which halothane half-maximally activates SERCA1. SDS gel electrophoresis of labeled membranes indicates that 38-56% of [14C]halothane incorporates into SERCA1, and 38-53% in lipids. Distribution of label among the three fragments produced by controlled tryptic digestion of SERCA1 suggests heterogeneous halothane binding presumably in discrete sites in the enzyme. The results provide the first direct evidence that halothane binds to SERCA1. Potentially this binding could be related to anesthetic effect on enzyme's function.
AB - The activity of various Ca2+-ATPases is affected by volatile anesthetics, such as halothane, commonly used in clinical practice. The effect on the enzyme in skeletal muscle sarcoplasmic reticulum (SR) is biphasic, including stimulation at clinical anesthetic concentrations and subsequent inhibition at higher concentrations. We have previously proposed that the action of a volatile anesthetic on Ca2+-ATPases results from its binding in the interior of the enzyme molecule [Lopez, M.M. and Kosk-Kosicka, D. (1995) J. Biol. Chem. 270, 28239-28245]. Presently, we investigated whether the anesthetic interacts directly with the skeletal muscle SR Ca2+-ATPase (SERCA1) as evidenced by binding. Photoaffinity labeling with [14C]halothane demonstrated that the anesthetic binds saturably to SR membranes, and that ~80% of the binding is specific, with a K(I) of 0.6 mM. The K(I) value agrees well with the concentration at which halothane half-maximally activates SERCA1. SDS gel electrophoresis of labeled membranes indicates that 38-56% of [14C]halothane incorporates into SERCA1, and 38-53% in lipids. Distribution of label among the three fragments produced by controlled tryptic digestion of SERCA1 suggests heterogeneous halothane binding presumably in discrete sites in the enzyme. The results provide the first direct evidence that halothane binds to SERCA1. Potentially this binding could be related to anesthetic effect on enzyme's function.
KW - Halothane
KW - Photoaffinity labeling
KW - SERCA1
KW - Tryptic digestion
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U2 - 10.1016/S0014-5793(96)01526-8
DO - 10.1016/S0014-5793(96)01526-8
M3 - Article
C2 - 9037193
AN - SCOPUS:0031041062
SN - 0014-5793
VL - 402
SP - 189
EP - 192
JO - FEBS Letters
JF - FEBS Letters
IS - 2-3
ER -