A Study on the Motion Of Protenis in Excitable Membrane Fragment by Nanosecond Fluorescence Polarization Spectroscopy

Abstract

The motion of two fluorescent dyes, 1 -anilino-8-naphthalene sulphonate, which binds reversibly, and 5-dimethylamino-l-naphthalene sulphonyl chloride, which binds covalently to excitable membrane fragments in vitro, has been studied by nanosecond fluorescence polarization spectroscopy. Both dyes were strongly immobilized by their association with membrane proteins. Solubilization by Triton X-100 of an important fraction (60°/0) of the proteins from membrane fragments heavily labelled with 5-dimethylamino-i -naphthalene sulphonyl chloride was accompanied by a dramatic increase of motion. It is concluded that proteins are strongly immobilized within the membrane phase.For years, the phenomenon of membrane excitability has been almost exclusively studied by the convenient, but necessarily limited techniques of electrophysiology [ i]. The recent developments of new physical and chemical methods have shed light upon the whole field and a molecular approach to this essential physiological mechanism now becomes feasible. I n particular, fluorescence spectroscopy appears to be especially convenient and versatile since it provides deep insight on the structure, interaction and motion of macromolecules both dispersed in solutions or integrated in a biological membrane A n important improvement of this last technique comes from the utilisation of nanosecond light pulses. By the measurement of the polarization of the fluorescent light emitted as an explicit function of time in the nanosecond range [6,[14][15][16][17]22,23], the estimation of the rotational motion of molecules labelled by convenient fluorophores becomes possible.We have carried out a nanosecond fluorescence polarization study of membranes prepared from the electric organ of the eel Electrophorus electricus. These membrane fragments derive from the innervated, and thus excitable surfaces of the electroplaxes, the elementary units of the electric organ. As recently shown by Kasai and Changeux[18] these purified membrane fragments are still excitable by cholinergic agonists in vitro, and thus constitute an exUnusual Abbreviations. Anilino naphthalene sulphonate, l-anilino-%naphthalene sulphonate; dansyl chloride, 5-dimethylamino-l-naphthalene sulphonyl chloride; Triton X-100, p-isocetylpolyoxyethylenephenol polymer.[2 -17,20 -291.Enzyme. Acetylcholine esterase (EC 3.1.1.7). cellent material for the study of chemical excitability a t the molecular level. We shall be concerned, in this first paper, with the motion of proteins in excitable membranes at rest. I n subsequent papers, we shall deal with the motion of membrane proteins during excitation.We have used two different fluorescent probes: l-anilino-%naphthalene sulphonate, which reversibly binds to the membrane fragments [12,13], and 5-dimethyl amino-1-naphthalene sulphonyl chloride (dansyl chloride) which attaches covalently to proteins from the membrane fragments [13]. With both probes it is shown that membrane proteins are strongly immobilized when integrated in the membrane structure and that the solubi...

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Allosteric Receptors: From Electric Organ to Cognition

“…The results were disappointing: no specific responses to nicotinic ligands were recorded. The in vivo measurements monitored the membrane potential, and the in vitro measurements monitored the average states of membrane proteins (e.g., their motion) (118). A visit to Manfred Eigen in 1969 in Göttingen, whom I personally knew for the work he had presented at the Pasteur Institute…”