Abstract
Previous work showed widespread saturable binding of halothane in rat brain. To determine whether this represents selective binding to a few widespread proteins or less selective binding to many different proteins, we used [14C]halothane photolabeling and quantitative electrophoresis/autoradiography in rat cerebellar homogenates. Many proteins incorporate label. Stoichiometry values ranged from 0 to 4 at 0.2 mM [14C]halothane in a group of 24 randomly selected protein bands. Apparent IC50 values from unlabeled halothane competition experiments ranged from 0.2 to 2.0 mM, with soluble protein having significantly lower values (higher affinity) than membrane protein. Chloroform inhibited halothane labeling similar to unlabeled halothane but with higher apparent IC50 values, whereas isoflurane and an anesthetic, cyclobutane (1-chloro-1,2,2-trifluorocyclobutane), inhibited halothane labeling to a smaller degree. A nonanesthetic, cyclobutane (1,2-dichlorohexafluorocyclobutane), inhibited halothane labeling the least. We conclude that halothane binding motifs are sufficiently degenerate to be found in many proteins, both soluble and membrane-bound.
Footnotes
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This work was supported by National Institute of General Medical Sciences Grants 51595 and 55876.
Abbreviations
- PBS
- phosphate-buffered saline
- PAGE
- polyacrylamide gel electrophoresis
- RD
- reflective densities
- OD
- optical densities
- PDB
- Protein Data Bank
- F3
- 1-chloro-1,2,2-trifluorocyclobutane
- F6
- 1,2-dichlorohexafluorocyclobutane
- Received July 12, 2001.
- Accepted October 9, 2001.
- The American Society for Pharmacology and Experimental Therapeutics
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