In contrast to previous conformational studies with rabbit skeletal and cardiac tropomyosins, (i) when the cysteine side chains of chicken gizzard tropomyosin were reacted with 5,5'-dithiobis(2-nitrobenzoate), an interchain disulfide cross-link was not produced, (ii) when they were labeled with pyrenylmaleimide , excimer fluorescence was not observed, and (iii) when they were labeled with didansylcystine , a long-lived fluorescence component did not appreciably contribute to the fluorescence decay over a large temperature range including the major unfolding transition. In addition, the temperature dependence of the ellipticity at 222 nm did not reveal a pretransition prior to the main helix unfolding transition. This indicates that gizzard tropomyosin does not exhibit a localized chain-open state in the region of its cysteine residues, analogous to that seen with cardiac and skeletal tropomyosins, nor in any other region of the molecule. As a consequence, these observations suggest that gizzard tropomyosin is more rigid than striated tropomyosin.
A B s T R A c T The effects of liquid fluorocarbons as bathing media were determined by use of in vitro neuromuscular preparations. Rat hemidiaphragms were bathed in either oxygenated fluorocarbon (FC) emulsion or standard oxygenated Krebs solution. Contractile force in response to simple supramaximal nerve stimuli as well as to high frequency stimulation was greater, while twitch:tetanus ratio was smaller in FC emulsion. With such medium, post-tetanic potentiation of contraction was also more consistently observed. Indirectly stimulated diaphragms survived longer in FC emulsion. After cessation of oxygenation, oxygen tension (pO2) of the medium declined more rapidly with Krebs than with FC emulsion; pO2 directly correlated with force of contraction. Similarly, in the chick biventer cervicis preparation, FC emulsion enhanced nerve-stimulated force of contraction; returning the preparation to standard Krebs solution reversed this phenomenon. Dose-response curves of muscle contraction in response to acetylcholine and KC1 administration were shifted upward during FC emulsion superfusion. Frequency of miniature endplate potentials was lower in FC emulsion than that observed in Krebs solution, measured from the same cell of the rat diaphragm. Resting membrane potentials were also greater in muscle cells sampled from FC emulsion-bathed preparations. These data suggest that FC emulsion is superior to standard Krebs solution as a bathing medium for in vitro neuromuscular preparations by virtue of the high solubility of oxygen in it.
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