ATP Muscle Lab

Introduction: Muscle contraction caused by ATP is one of the most dramatic physiological phenomena that can be demonstrated. ATP or adenosine triphosphate, is the basic biochemical energy source in living organisms. Skeletal muscle is used for this demonstration due to its relatively large striations which make the effects of ATP readily observable. Rabbit muscle is preferred because it is readily obtained and excised. Preparation of the psoas involves separating small bundles of fibers from the main body of the muscle, tying the bundles to wooden rods, cutting them free and them storing them in a very cold glycerol solution. The bundles are tied before being cut from the main muscle in order to prevent contraction. The glycerol solution prevents the freezing of the muscle myofibers at the low temperatures required for storage. Further, the glycerol does not appreciably affect the concentration of the auxiliary enzymes necessary for contraction such as the phosphorpheases and myokinase. The general procedure for causing muscle contraction is to add several drops of an ATPKCl-MgCl2 solution to several fibers. The biochemical reactions among the ATP, metal ions, and enzymes present in the muscle provide the energy that causes the protein filaments in the fibers to draw together and contract the fibers. Materials: 1 vial (solution A) 0.25% ATP solution in distilled water 1 vial (solution B) 0.25% ATP solution plus 0.05M KCl plus 0.001M MgCl2 in distilled water 1 vial (solution C) 0.05M KCl plus 0.001M MgCl2 in distilled water 1 tube rabbit psoas muscle in glycerol sharp scissors teasing needle Petri dish forceps, very-fine point dropper pipet microscope slides and cover slips ruler, millimeter scale microscopes, compound and dissecting Procedure: 1. Remove the rod with the psoas muscle from the glycerol. Pour the glycerol into a Petri dish. 2. Using the scissors, cut the muscle into 2cm length pieces. Place the pieces in the glycerol solution in the Petri dish. One 2cm piece should yield sufficient material for each student or team of students. 3. The thinner the groups of myofibers are, the greater will be the contraction demonstrated. This is because the uncontracted muscle fibers in the center of a thick bundle inhibit the contraction of the outer myofibers. Therefore, very thin bundles of fibers or ideally, single fibers should be teased from the larger muscle segment. 4. Muscle fiber microanatomy can be observed by preparing a wet mount of one of the bundles or single fibers using glycerol. The rabbit psoas myofibers observed under low and high power magnification exhibit clearly defined striation typical of skeletal muscle. The following steps may be done using one or several strands. If sufficient amount of material is available, several strands should be observed. 5. Measure the strand in a wet mount using a millimeter scale and record. The measurement provides a point of reference for the degree of contraction observed.

6. Flood the fiber with several drops of ATP metal salts solution (solution B) and observe the action of the fiber. 7. After about 30-45 seconds, remeasure the fiber and calculate the degree of contraction. Was the length of the fiber the only dimension that changed? 8. Prepare a wet mount of one of the contracted fibers and observe with a compound microscope. Describe any differences that you can observe between a relaxed and contracted fiber. 9. Try different combinations of the ATP-only solution (solution A), metal salts-only solution (solution C), and the ATP plus metal salts solution (solution B) on fresh myofibers. Determine what substances are necessary for muscle contraction to occur. Assignment: 1. Carefully record all your methods and data. 2. Sketch and label skeletal muscle tissue. 3. Explain how skeletal muscle contracts and relate what you know to your findings in this lab. Include the roles of Ca2+ ions, troponin, tropomyosin, actin, myosin, cross-bridge formation, movement and breakage, and ATP.