The orientation of the SH-proteins in muscle and nerve tissues was analyzed by measuring the angular distribution of fluorescence depolarization of the dyes labeled on the samples, using a novel fluorescent thiol reagent, N- (1-anilinonaphthyl-4) maleimide (ANM) together with 1,8-anilinonaphthalene sulfonate (ANS) as a hydrophobic probe.
1. The fraction of the fluorescence intensity of the oriented components, orientation ratio (Ro), was approximately 20% in ANM-labeled samples and 17% in ANS-labeled ones regardless of the kind of samples ; rabbit muscle fiber bundles, glycerinated muscle fibers, frog sartorius muscles and frog N. ishiadicus. When the samples were dried on air, the values increased slightly in ANM-labeled samples and considerably in ANS-labeled ones.
2. The Ro values of muscle fibers was slightly elevated by soaking in glycerine at -20°C, for several months, while that of fresh ANM-stained fibers increased after one day and then decreased by the same treatment. Fresh frog nerve was not stained by ANS, but an oriented fraction of about 7% appeared after glycerination. The fraction further increased by drying. The Ro values of ANM-labeled muscle fibers increased until 60 sec after the addition of trypsin and then decreased.
3. No oriented fraction was observed in the ventral cord of
Cambaras clarkii.
4. All of the samples showed symmetrical biaxial orientation which was parallel and perpendicular with the axis of the fibers. This mode was not essentially altered by drying.
5. In the most of the fresh samples, the parpendicular fraction was larger than the parallel one. The difference between the two fractions increased by drying, while it decreased by the treatment with glycerine.
The present experimental results clearly show that a part of the SH-proteins is oriented on the surface of these fibers.
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