ABSTRACT
Protein kinase C (PKC) is a signal transducing protein that has been implicated in binding alcohol and anesthetics. The alcohol and anesthetic binding of protein kinase C delta C1B domain has been determined previously by photolabeling and mass spectrometry [J. Das, G.H. Addona, W.S. Sandberg, S.S. Husain, T. Stehle, K.W. Miller, Identiffcation of a general anesthetic binding site in the diacylglycerol-binding domain of protein kinase C delta, J. Biol. Chem. 279 (2004) 37964-37972]. Here we studied photoincorporation of 3-azioctanol, a photoactive analog of octanol into PKC delta C1B in two buffer systems containing tris and hepes. The extent of photoincorporation was higher in hepes compared to tris as determined by high performance liquid chromatography and mass spectrometric analysis. The results are explained on the basis of the presence of number of primary hydroxyl and amino groups in tris and hepes molecules that could affect the binding of alcohol molecules to protein. This observation will be useful in selecting buffer system for biochemical studies on PKC.
Subject(s)
HEPES/chemistry , Octanols/radiation effects , Protein Kinase C-delta/chemistry , Tromethamine/chemistry , Anesthetics/chemistry , Binding Sites , Buffers , Chromatography, High Pressure Liquid , Octanols/chemistry , Protein Structure, Tertiary , Spectrometry, Mass, Electrospray Ionization , Time FactorsABSTRACT
To overcome the difficulties of locating the molecular sites of general anesthetic action, we synthesized a novel photoactivable general anesthetic, 3-(2-hydroxyethyl)-3-n-pentyldiazirine (3-diazirinyloctanol), which anesthetized tadpoles with an ED(50) of 160 microM. Subanesthetic concentrations of 3-diazirinyloctanol enhanced GABA-induced currents in GABA(A) receptors, an effect that has been implicated in general anesthetic action. It also enhanced [(3)H]muscimol binding to this receptor. In muscle nicotinic acetylcholine receptors (nAcChoR), it inhibited the response to acetylcholine with an IC(50) of 33 microM. 3-Diazirinyloctanol's pharmacological actions were comparable to those of octanol. 3-(2-Hydroxyethyl)-3-[4,5-(3)H(2)]-n-pentyldiazirine photoincorporated into Torpedo nAcChoR-rich membranes mainly in the alpha subunit with 70% being in a proteolytic fragment containing the M4 transmembrane segment. Agonist enhanced the photolabeling 10-fold in a fragment containing the M1, M2, and M3 transmembrane segments. Thus, 3-diazirinyloctanol is a novel general anesthetic that acts on, and can be photoincorporated into, postsynaptic receptors.
