INTERACTION BETWEEN TITIN AND α-ACTININ

The interaction of titin with a major Z-band protein, 0:-actinin was investigated by a solid phase binding assay and a binding study. Native titin was biotinylated and allowed to react with myofibrils or microtiter wells coated with a-actinin. The bound biotinylated titin was detected by the avidin-biotin-peroxidase method. Biotinylated titin bound to a-actinin in a concentration-dependent manner. Unlabeled titin apparently competed with biotinylated titin on the binding site(s) of a-actinin. The possible binding site on the titin for 0:-actinin was also examined by the analysis of affinity column-purified titin fragments with a monoclonal anti-titin antibody. It appeared that a-actinin had binding site(s) in the A-band domain of the titin. In both intact myofibrils and I-Z-I segments, biotinylated titin labeled the Z-bands. These results confirm the interaction of titin and a-actinin in situ. Our finding that titin can bind to the Z-band, probably through its interaction with a-actinin, supports a role of titin in integrating the A-bands and I-Z-I segments into sarcomeres. Titin (connectin) filaments span from the Z-band to the M-line, running between thick and thin filaments (2, ll, l6). Biochemical and electron microscopic studies have shown that titin interacts with myosin filaments and two M-line proteins (6, l0, l2). Several studies have demonstrated the possible anchorage of titin to the Z-band. The titin epitope at the A-I junction moves to the Z-bands after dislodging titin from the A-band or the M-line structure (l7). Furthermore, Fiirst er ai. (2) reported two monoclonal anti-titin antibodies which recognized titin epitopes close to the Z-band. These data suggest that one end of the titin molecule is attached to the Z-band region. Titin filaments seem to attach to the I-Z-I segments via the Z-band specific end of the molecule in the early development of myofibrils (3, l4). However, the study of the interaction between titin and a-actinin, a structural backbone of Z-band, has not yet been reported. The present study was designed to investigate the interaction between titin and a-actinin. Titin II (a proteolytic product of titin I) was biotinylated and used in a solid-phase binding assay or in the binding study of titin in the myofibrils. The binding of biotinylated titin to 0:-actinin coated on the microtiter wells or the sarcomeric structures was detected by the avidin-biotin-peroxidase method. MATERIALS AND METHODS Purification of Native Tiiin and a-Actinin Native titin and a-actinin were purified from chicken breast muscle as described by Kimura er ai. (6) and Langer and Pepe (9), respectively. Purified protein was labeled with biotin. The conjugated protein was separated from free biotin by gel filtration on Sephadex-G 25 column. Solid-Phase Binding Assay Microtiter plates were coated with purified protein at 10 jag/ml. After washing and blocking with 1% bovine serum albumin in phosphate-buffered saline (PBS), the wells were incubated with biotinylated protein at different concentrations in PBS at 37°C for lh. The wells were washed with PBSTween (0.059/0 Tween in PBS) and then incubated with a 1:500 diluted avidin-biotin-peroxidase mixture (Vector Laboratory, CA, U.S.A.) for l h. After washing with PBS-Tween and then PBS, 100 ,al of substrate solution (2 mg of 2,2’-azino-di-3-ethylbenzthiazoline sulfonate, l0 til of 30% H202 in