Preparation of the muscle fibers and actomyosin

INSTRUCTOR PREPARATION OF GLYCERINATED MUSCLE:

1. Inject 0.5 mg. curare per kilo into the rat or rabbit. Decapitate it and, after draining blood from the carcass, cool it in the refrigerator for about 10 minutes. Eviscerate it and expose the psoas muscle; this muscle lies along the ventral aspect of the group of trunk muscles which run below the spinal column. Most of the psoas muscle should be removed from the body immediately and put into a beaker chilled with ice. This will be used for the preparation of the actomyosin threads. The muscle remaining in situ will be used to prepare the glycerinated fibers.

2. Preparation of glycerinated fibers: With glass needles strip bundles of fibers 2 to 5 mm in diameter from the muscle and tie them to wooden applicator sticks or glass rods with thread. Place the bundles in a 50% solution of glycerol in water, previously cooled to 0^oC. Several bundles can be placed in one test tube. Place the test tubes containing this preparation in the ice box at 0^oC for a period of 24 hours. Decant the old glycerol and add fresh 50% glycerol. Place the preparation in the freezing compartment of the refrigerator for at least a week.

STUDENT EXPERIMENTS:

Transfer the glycerinated fiber bundles to 25% glycerol at 0^oC and let them stand for 30 minutes. Tease out a single fiber about 15 mm long and 1 mm thick, and transfer to ice cold 0.05 M phosphate buffer at pH 7.0

Place it on a clear plastic ruler and measure its original and final length after each of 5 additions as indicated in the following table for protocols I to IV.  Use separate fibers to study the effects of each protocol:

Examine fibers in different states-

• - do your major observation with your disecting microscope
• - some time during the period observe fibers:
• - under the compound light microscope [low (10x) and medium (43x) power objectives]
      - with varying light conditions.
      - with polarized light.
• - under the phase contrast microscope.

INSTRUCTOR PREPARATION OF ACTOMYOSIN SOLUTION:

3.  Preparation of actomyosin threads: weigh the (previously weighed) chilled beaker which now contains the larger part of the psoas muscles. Add 3.0 ml. of Guba-Straub solution for each gram of muscle, and mince in the blender until a fine suspension is obtained, usually in about one minute. Allow the suspension to stand in the refrigerator for one hour. Centrifuge; the supernatant layer contains the actomyosin. Draw off some of the supernatant layer; if it is too viscous, dilute with Guba-Straub solution.

STUDENT EXPERIMENTS:

4. Artificially reconstituted actomyosin threads.  Prepare a petri dish containing 0.05 M KC1 and  0.001 M MgCl₂, or cold distilled water.  Draw into a tuberculin syringe some of the supernatant fluid from the centrifugation and allow it to flow into the finger bowl by slowly expelling solution from the needle tip while waving it, immersed in the salt solution. If the fluid is too viscous to expel from the syringe, more dilution may be required.

Allow the fibers to stand in the salt solution ~10 minutes then use your glass needles to place one on the plastic ruler and observe with the disecting microscope.

Using the same procedure as for the glycerinated fibers, study the behavior of the actomyosin threads when one drop of ATP is added in one of the effective protocols from the protocol matrix above.

5.  KCl dependence of Superprecipitation. Add actomyosin, distilled water and 0.6 M KCL in the proportions stated for the 4 experiments A-D in Table II.  Record the initial absorbance at 540 nm.  Then to each cuvette add 0.1 ml of 0.1 M Mg Cl₂ and 0.05 ml of 0.1 M ATP.  Again, record the absorbance after the reading has stabilized.  Stir by inversion with parafilm at intervals to encourage an even reaction.  Plot your data as bar plots.

6.  Gedanken Experiment.  Design an experimental protocol for examining the role of ATP and divalent cations in controling the superprecipitation experiment.  If you have the time and materials carry out the experiment.