The selectivity of lipid-protein interaction for spin-labeled phospholipids and gangliosides in nicotinic acetylcholine receptor-rich membranes from Torpedo marmorata has been studied by ESR spectroscopy. The association constants of the spin-labeled lipids (relative to phosphatidylcholine) at pH 8.0 are in the order cardiolipin (5.1) approximately equal to stearic acid (4.9) approximately equal to phosphatidylinositol (4.7) > phosphatidylserine (2.7) > phosphatidylglycerol (1.7) > G(D1b) approximately equal to G(M1) approximately equal to G(M2) approximately equal to G(M3) approximately equal to phosphatidylcholine (1.0) > phosphatidylethanolamine (0.5). No selectivity for mono- or disialogangliosides is found over that for phosphatidylcholine. Aminated local anesthetics were found to compete with spin-labeled phosphatidylinositol, but to a much lesser extent with spin-labeled stearic acid, for sites on the intramembranous surface of the protein. The relative association constant of phosphatidylinositol was reduced in the presence of the different local anesthetics to the following extents: tetracaine (55%) > procaine (35%) approximately benzocaine (30%). For stearic acid, only tetracaine gave an appreciable reduction (30%) in association constant. These displacements represent an intrinsic difference in affinity of the local anesthetics for the lipid-protein interface because the membrane partition coefficients are in the order benzocaine >> tetracaine approximately procaine.

We have examined the immunoreactivity of acetylcholinesterase from different vertebrate species with a rabbit antiserum raised against the purified rat brain hydrophobic enzyme (G4 form). We found no significant interaction with enzymes from Electrophorus, Torpedo, chicken, and rabbit. The antiserum reacted with acetylcholinesterases from the brains of the other mammalian species studied, with titers decreasing in the following order: rat = mouse greater than human greater than bovine. The serum was inhibitory with murine and human acetylcholinesterases, but not with the bovine enzyme. The inhibition was partially depressed in the presence of salt (e.g., 1 M NaCl). In those species whose acetylcholinesterase was recognized by the antiserum, both soluble and detergent-soluble fractions behaved in essentially the same manner, interacting with the same antibodies. The apparent immunoprecipitation titer was decreased in the presence of salt, and it did not make any difference whether NaCl was included in the solubilization procedure or added to the extracts. Both G1 and G4 forms of acetylcholinesterase in the soluble and detergent-soluble fractions were recognized by the antiserum, and in the case of the human enzyme, by monoclonal antibodies produced against human erythrocyte acetylcholinesterase. However, the monomer G1 showed a clear tendency to form smaller complexes and precipitate less readily than the tetramer G4. Although we cannot exclude the existence of significant differences between the various molecular forms of acetylcholinesterase, our results are consistent with the hypothesis that they all derive from the same gene or set of genes by posttranslational modifications.

Fluorescent probes have been selectively introduced into skeletal muscle myosin subfragment-1 and the fluorescence emission characteristics of the labeled products studied. The fluorophores employed were the thiol-specific reagents N-[[(iodoacetyl)aminolethyl-5-naphthylamine-1-sulfonic acid and 5-(iodoacetamido)fluorescein, the spectral properties of which render them a particularly effective donor-acceptor pair in Förster energy-transfer studies. Alkali 1 light chain, labeled at a single cysteine with either of these probes, was incorporated into chymotryptic subfragment-1 by the exchange procedure of Wagner & Weeds [Wagner, P.D., & Weeds, A.G. (1977) J. Mol. Biol. 109, 455-473]. The resultant, fluorescently labeled subfragment-1 was isolated by ion-exchange chromatography. Determination of the extent of incorporation by extinction and fluorescence indicated that greater than 80% of the subfragment-1 population possessed a fluorescently labeled alkali 1 light chain. The introduction of labeled alkali 1 did not perturb the K+-, Ca2+-, or actin-activated adenosine triphosphatases of subfragment-1. The addition of adenosine triphosphate (ATP), liganded by various cations, to this singly labeled subfragment-1 induced a 6-10% decrease in the fluorescence intensity of the extrinsic chromophore. An intensity decrease of approximately 4% was obtained when the hydrolysis of ATP was complete, and also upon direct addition of adenosine diphosphate. The ATP analogue adenylyl imidodiphosphate induced a decrease of approximately 7% in intensity. The addition of F-actin to the subfragment-1 in the presence of MgATP elicited no further fluorescence intensity change. A second, appropriate fluorophore was introduced into the singly labeled subfragment-1 at the SH1 thiol on the heavy chain. Förster energy transfer was observed between this labeled site and the fluorophore previously introduced on the alkali 1 light chain. The measured efficiency of energy transfer indicated that the two fluorophores were approximately 40 A apart. The same value was obtained upon reversal of the donor and acceptor attachment sites, suggesting that the uncertainty in the calculated distance introduced by the choice of orientation factor is probably less than 20%. Steady-state observations did not reveal any obvious change in this distance upon the addition of MgATP and then F-actin to the doubly labeled subfragment-1.

No abstract

The binding of A D P to heavy meromyosin. and the separated subfragment 1 components S-1 ( A l ) and S-1 (A2), has been observed by ultraviolet spectrophotometry. The results are compatible with the presence of spectroscopically equivalent and independent sites, one per head, a t both 10 'C and 25 ' C. We d o not observe the heterogeneity of binding and of the spectroscopic response that has been reported. The binding has also been followed by other methods sensitive to the effect of ligand on the aromatic residues of the protein, viz. intrinsic fluorescence of heavy meromyosin and changes in the near-ultraviolet Cotton effects of myosin, and its active fragments. Within the limits of our experimental precision, the binding profiles. based on concentration of myosin heads, are the same for myosin as for subfragment 1. A perturbation in the circular dichroism is also generated by pyrophosphate, which competes with A D P . The spectra suggest that subsites for the purine ring and the diphosphate can be recognised. The sensitivity of binding profiles obtained by methods of the kind used here to cooperative o r antagonistic interactions between the binding sites has been analysed. It is clear that sizeable effects of this nature could be concealed by the bindingcurves, even for high experimental precision.The binding of A D P to myosin and its active fragments has been studied by a variety of methods. Direct measurements. mostly on heavy meromyosin. by equilibrium dialysis or gel partition and in the presence of magnesium ions. have led to results compatible with the existence of two independent and equivalent binding sites per molecule with association constants in the vicinity of lo6 M '. depending somewhat o n the temperature and ionic strength [l -41. The limitations of experimental precision are such that a considerable degree of heterogeneity of binding, arising for example from cooperativity, could be accommodated by the data. The only direct assertion of such complexity conies from Morita and coworkcrs, and is based on a spectropliotometric method of following the binding process. Morita [S] first discovered that the association of ATP, A D P or pyrophosphate with the active centre generated a perturbation in the ultraviolet absorption spectrum of the protein, that could be observed as a difTcrence spectrum. Its magnitude moreover was reported [5,6] to reach its maximum value at 25 ' C with saturation of only half the binding sites. At 8 'C, on the other hand. the inaximuni difference spectrum is developed only on saturation of all sites, though its amplitude remains the same. The interpretation placed on the results is that only one of the lwo myosin heads produces a spectroscopic response, and that at 25 'C the site on this head has a vastly greater affinity for ADP. At 8 ' C the affinity of the 'silent' site becomes comparable t o that of the other. Moreover subfragment 1 was reported to have sites of uniformly high affinity in all conditions, but only half the molecules were responsible for the spectr...