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Method for isolation of intact titin (connectin) molecules from Mammalian cardiac muscle

I. M. Vikhlyantsev1*, A. D. Okuneva1,2, U. V. Shumilina1, N. N. Salmov1, A. G. Bobylev1, N. V. Molochkov1, and Z. A. Podlubnaya1,2

1Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, 142290 Pushchino, Moscow Region, Russia; fax: (4967) 33-0553; E-mail: vikhlyantsev@iteb.ru

2Pushchino State Institute of Natural Sciences, pr. Nauki 3, 142290 Pushchino, Moscow Region, Russia

* To whom correspondence should be addressed.

Received November 27, 2012; Revision received January 20, 2013
Cardiac titin was isolated from rabbit and ground squirrel ventricular muscles by a method that was used earlier to obtain myofibrils with intact minor proteins located in A-bands of sarcomeres (Podlubnaya, Z. A., et al. (1989) J. Mol. Biol., 210, 655-658). Small pieces of cardiac muscle were incubated for 2-3 weeks at 4°C in Ca2+-depleting solution before their homogenization to decrease activity of Ca2+-dependent proteases. Then the muscle was homogenized, and titin was isolated by the method of Soteriou, A., et al. (1993) J. Cell Sci., 14, 119-123. In control experiments, titin was isolated from cardiac muscle without its preincubation in Ca2+-depleting solution. Sometimes control titin preparations contained only T2-fragment, but generally they contained ~5-20% N2B-isoform of titin along with its T2-fragment. Preparations of titin obtained from rabbit cardiac muscle by our method contained ~30-50% of N2BA- and N2B-titin isoforms along with its T2-fragment. The content of α-structures in titin isolated by our method was increased. Actomyosin ATPase activity in vitro increased in the presence of titin preparations containing more intact molecules. This result confirms the significant role of titin in the regulation of actin–myosin interaction in muscles. The method used by us to preserve titin might be used for isolation of other proteins that are substrates of Ca2+-dependent proteases.
KEY WORDS: striated muscles of mammals, NT-, N2A-, N2BA- and N2B-isoforms of titin, T2-fragments, actin-activated ATPase activity of myosin, Ca2+-depleting solution

DOI: 10.1134/S0006297913050039