CORNELL BIOPL 4440 - Chapter 10 Actin and microfilament-mediated processes

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Chapter 10. Actin and microfilament-mediated processes 10.1. Discovery of actomyosin, and the mechanism of muscle movementMovement is one of the most easily distinguished characteristics of life. Theodor Engelmann (1879) noticed all kinds of motion in plants and protozoa, including amoeboid movement and cytoplasmic streaming (Figure 10-1). He suggested that these activities may be a primitive version of the specialized movements that occur in muscle and indeed, the same molecular mechanisms may be involved in them all. Seventy years later, Albert Szent-Györgyi (1949b) put it this way: “All living organisms are but leaves on the same tree of life. The various functions of plants and animals and their specialized organs are manifestations of the same living matter. This adapts itself to different jobs and circumstances, but operates on the same basic principles. Muscle contraction is only one of these adaptions.” If all life shows motion, which cell, tissue, organ or organism shall we choose to study in order to unravel the mysteries that underly the vital processof movement in living organisms, and to give us the most clear and profound answers? Szent-Györgyi (1948) suggests that we use the cells that are most specialized for movement: skeletal muscle. The excitement of some of the pioneers in muscle research has been captured in their published lectures and monographs (Szent-Györgyi, 1947,1948,1953; Mommaerts, 1950b; Weber, 1958; Huxley, 1966,1969,1996; Huxley, 1980; Straub, 1981; Engelhardt, 1982).While most biochemists in the 1930s were studying water soluble enzymes, the husband and wife team of Vladimir Engelhardt and Militza Lyubimova violated one of the canons of biochemistry, and studied the “residue instead of the extract” (Engelhardt, 1982). In those days, following the acceptance of Sumner’s (1926) work, the residue was thought to be composed of mundane structural proteins and not exciting enzymes. But, while studying muscle, Engelhardt and Ljubimowa (1939) found that myosin, a “structural” protein that had previously been isolated from muscle by Wilhelm Kühne (1864), was also an enzyme capable of hydrolyzing ATP.339Szent-Györgyi became interested in muscle after he read about the ATPaseactivity of myosin. He thought that it may be the mechanochemical transducerthat coupled the chemical energy of ATP to the mechanical energy of contraction, and he set out to test his hypothesis. Realizing that he was standing on the shoulders of giants, Szent-Györgyi repeated the work of the “old masters” and isolated myosin using the method of Engelhardt and Ljubimowa (Szent-Györgyi and Banga, 1941). He extracted the muscle for anhour with an alkaline 0.6 M KCl solution to get the typical syrupy myosin preparation. He then prepared threads of myosin and put them on a slide and watched them under a microscope. Then he added ATP to the slide and mirabile dictu: they contracted! It was as if he had seen life itself!Ilona Banga continued to isolate myosin in Szent-Györgyi’s laboratory, buthad to go home early one day and left the minced muscle in KCl all night. Thenext morning they realized that the extract was thicker than the usual extract and it also contracted more vigorously upon the addition of ATP. They called the original extract myosin A and the thick extract myosin B. It turned out thatthe difference between the two extracts was that myosin A was extracted while the muscle still contained ATP, and myosin B was isolated after all the ATP had been hydrolyzed. Szent-Györgyi suggested that Ferenc Brunó Straub investigate the difference between the weakly contracting myosin A and the forceful myosin B (Straub, 1981). Straub postulated that myosin B was enriched in a protein that was a contaminant in myosin A. Unbeknownst to Szent-Györgyi and Straub, the protein contaminant had been isolated by Halliburton in 1887 under the name myosin-ferment (see Finck, 1968). Straub extracted an ATP containing muscle with 0.6 M KCl, and then washed and dried the remaining muscle with acetone. The acetone powder was then extracted with water and a protein went into solution. This protein solution, when added to myosin A in the presence of ATP, caused the myosin to contract. Straub named this proteinactin, because it had the ability to act (Moss, 1988) and they renamed myosin B, actomyosin. Actin had the ability to activate the ATPase activity of myosin by about ten-fold, in addition to being able to cause the actomyosin mixture tocontract.Szent-Györgyi resurrected an earlier proposal by Karl Lohmann (Meyerhof, 1944), the discover of ATP, that the chemical energy of ATP provided the energy for muscle contraction, and moreover, that muscle 340contraction was essentially due to the interaction of actomyosin and ATP. However this conclusion was not widely accepted for a number of reasons, one of which, was, that the magnitude of the free energy released by the measured amount of ATP hydrolyzed, was insufficient to account for the workperformed by the contracting muscle (Mommaerts and Seraidarian, 1947; Perry et al., 1948; Hill, 1949; Mommaerts, 1950a; Szent-Györgyi, 1963; Gergely, 1964).Szent-Györgyi (1949a) decided to demonstrate beyond a shadow of a doubt that ATP provides the chemical energy for contraction. He and Varga (1950) developed a glycerinated muscle preparation. They extracted the muscle with 50% glycerol at low temperatures to make a permeabilized cell model (Arronet, 1973). Then, upon addition of ATP, the model contracted and developed the same tension as if it were an intact muscle. Contraction is thus due to the conversion of the chemical energy of ATP to the mechanical energy of muscle contraction. The inability to detect the relationship between free energy release from ATP and work was due to the fact that the magnitude of ATP hydrolyzed by a contracting muscle was underestimated since, in muscle,ATP is constantly being regenerated through a creatine phosphate system.From the first observation of the contraction of actomyosin threads under the microscope, Szent-Györgyi (1948) believed that the proteins themselves contracted. However structural data, which included x-ray diffraction images, as well as polarization, interference and electron microscopic images obtainedby Jean Hanson, Hugh Huxley and Andrew Huxley, indicated that the contractile proteins were not contractile at all, but slide past each other when they effected the shortening of a


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CORNELL BIOPL 4440 - Chapter 10 Actin and microfilament-mediated processes

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