Removal and Reconstitution of Z-line Material in a Striated Muscle

Short papers submitted expressly for this section, reporting original and significant findings of immediate interest and judged to be acceptable without major revision, will be published within approximately three months. See inside back cover for details. Many detailed structural studies of the Z line have been reported (14, 13, 9, 16). Huxley (13) infers that tropomyosin is the principal component of the Z line. Evidence that tropomyosin is not confined to the Z line alone has been presented (8) and was previously suggested by Hanson and Lowy (10). If tropomyosin is located in the Z line in crystalline form and also associated with thin filaments in another form, then it would be necessary for two structural forms of the protein to exist in the myofibril. Cohen and Longley (5) have shown that two forms of tropomyosin can be obtained. Corsi and Perry (6) extracted both actin and tropomyosin and removed Z lines and I-band material. Several reports (2, 8, 20) have shown that Z lines can be removed by trypsin treatment. Ebashi and Kodama's (7) work indicated that both tropomyosin and troponin are sensitive to trypsin; however, these authors reported that 50% of the tropomyosin in myosin B was unaffected by their conditions of hydrolysis. This was explained by the hypothesis that tropomyosin bound to myosin B was more resistant to tryptic attack than "free" tropomyosin. Since tropomyosin in the myofibril is probably associated with another structural protein, it seems equally plausible that tropomyosin may exhibit a relatively higher resistance to hydrolysis by trypsin. The evidence that tropomyosin is the main component of the Z line was, therefore, not conclusive. Also, it has recently been proposed that a-actinine is a component of the Z line (4). We have studied the involvement of tropomyosin in the Z line by selective extraction of the myofibril followed by recombination of various protein fractions under defined conditions of ionic strength. It was reported (19) that tropomyosin depressed the Ca++-activated ATPase activity of reconstituted actomyosin, and we employed this observation to assay the protein fractions for tropomyosin. MATERIALS AND METHODS Rabbit psoas muscle which had been glycerinated for at least 30 days in the usual buffered (pH 7.0) 50% glycerol was the starting material. The tissue was teased into thin bundles (40-70) of myofibrils in 2 mM Tris pH 7.6, 1 m dithiothreitol at 0OC. After 1 hr, the teased myofibrils were transferred to fresh …