Volume 62, No. 3, 1997—JOURNAL OF FOOD SCIENCE—451 An Hypothesis Paper Skeletal Muscle Connectin Primary Structures as Related to Animal Species and Muscle Type R. TANABE, S. MUROYA, I. NAKAJIMA, K. CHIKUNI, and H. NAKAI ABSTRACT Differences in molecular weights and partial amino acid sequences of connectin(titin) were determined for cattle, pig and chicken skeletal mus- cles. Peptide mapping analysis results differed according to animal spe- cies. Amino acid sequences deduced from partial nucleotide sequences of connectin also differed according to animal species at immunoglob- ulin-like (Ig) and fibronectin type 3 (FN3) domains. In chicken, the mo- lecular weight of connectin from leg muscles was higher than that from pectoral muscles. Differences in meat texture and conditioning may re- late to connectin and extent of its breakdown. Key Word: connectin, titin, skeletal muscle, meat texture, meat condi- tioning INTRODUCTION CONNECTIN is an elastic protein of striated muscle initially re- ported by Maruyama et al. (1976) and also called titin (Wang et al., 1979). The apparent molecular mass of connectin is 3,000 kDa (Maruyama, 1994; Trinick, 1994). Each connectin molecule extends as a long (1 μm) filament from the Z disc to the M line in a sarcomere and comprises an elastic segment at the level of the I band and an inelastic segment at the level of the A band. Connectin filaments keep the thick filaments centered within the sarcomere during force generation (Horowits et al., 1986). During meat conditioning, the connectin molecule splits into two polypeptides at a point 0.34 μm apart from the Z disc (Tan- abe et al., 1994). The molecular mass of one peptide is 1,200 kDa (1,200 kDa-subfragment); the other peptide is called - connectin. In postmortem muscle, the sarcoplasmic calcium ions increase, and calcium causes connectin to split into two poly- peptides (Koohmaraie, 1994; Takahashi et al., 1992). This split- ting is very important for meat texture, because it lessens the elasticity of meat during conditioning (Takahashi and Saito, 1979). Connectin filaments split more rapidly in more tender meat (Anderson and Parrish, 1989; Huff-Lonergan et al., 1995). However, Fritz et al. (1993) concluded that connectin content did not distinguish ‘tough’ from ‘tender’ beef. The time required for conditioning varies according to animal species. Within meat conditioned quickly, splitting is also rapid (Anderson and Par- rish, 1989; Huff-Lonergan et al., 1995; Paxhia and Parrish, 1988). Differences in molecular structures of connectin may thus be a determinant of meat texture. Labeit and Kolmerer (1995) determined the complete cDNA sequence of human cardiac connectin and showed its structure consisted primarily of two main types of repeating domains (an immunoglobulin-like (Ig), a fibronectin type 3 (FN3)) and a PEVK domain. They proposed that PEVK domain primarily ac- Authors Tanabe, Muroya, Chikuni and Nakai are with the Dept. of Animal Products, and author Nakajima is with the Dept. of Animal Physiology, National Institute of Animal Industry, Ikenodai, Kuki- zaki-cho, Inashiki-gun, Ibaraki, 305, Japan. Direct inquiries to Dr. R. Tanabe. counted for the elasticity of the connectin molecule. They pro- posed that after extensibility of PEVK domain had been exhausted, the stable fold of the Ig domains would resist further extension. These domains may thus determine meat texture. The molecular structures of connectin in humans, rabbit and exper- imental animals have been studied extensively (Fritz et al., 1993; Jin, 1995; Labeit et al., 1990; Labeit and Kolmerer, 1995; Maruyama et al., 1994; Mu ¨ller-Seitz et al., 1993; Sebestyen et al., 1995). However, there are few reports on molecular structure of connectin in domestic animals. Our objective was to determine the differences in molecular weights of connectin and amino acid sequences of Ig, FN3 and PEVK domains in cattle, pig, chicken and muscle types. MATERIALS & METHODS Reagents Most reagents including S. aureus V8 protease (V8 protease) were purchased from Wako Pure Chem. Co. (Osaka Japan). Agarose for sep- arating DNA was from FMC Bio Products (Rockland, ME). Preparation and Ca treatment of myofibrils M. semimembranosus and M. longissimus thoracis were excised from cattle and pig, immediately after slaughter. M. pectoralis profundus, M. pectoralis superficialis, M. semitendinosus, M. semimembranosus, M. sartorius and M. bicepsfemoris were excised from chicken, immediately after slaughter. Myofibrils were prepared according to Perry and Grey (1956). To induce the splitting of connectin into 1,200 kDa-subfragment and -connectin in vitro, freshly prepared myofibrils were treated with 0.1 mM CaCl 2 as described by Takahashi et al. (1992). Peptide mapping Connectin was digested by V8 protease as described with modification (Cleveland et al., 1977). Connectin, -connectin and 1,200 kDa-subfrag- ment were separated by sodium dodecylsulfate gel electrophoresis (SDS- PAGE) according to Tatsumi and Hattori (1995). Each band containing 6 μg peptide was cut from the gel, which was then soaked in a solution with the same composition as that of the stacking gel (Laemmli, 1970). Each gel piece was placed in the well of the polyacrylamide gel (stacking gel:0.5% agarose, separating gel:12.5% acrylamide). V8 protease solu- tion containing 0.1 μg protease and 10% glycerol was added to the well. Electrophoresis was carried out at room temperature. When the marker dye reached the bottom of the stacking gel, electrophoresis was stopped for 25 min for digestion of the peptide by V8 protease. Electrophoresis was resumed to separate the peptides produced by V8 protease digestion. PCR amplification and sequencing of segments from the connectin gene Sequences of the oligonucleotide primers were based on the sequences of pig connectin reported by Fritz et al. (1993), because the sequence of cattle connectin gene is not known. The connectin molecule has Ig and FN3 domains. The sense primer for amplification of the Ig domain was 5'-CAGGTGGCTCCTTAAGGTTATTTGTTCC-3' and the antisense primer for the Ig domain was 5'-CAAAGGCAGACTTTGTTCCACT- GCTGTT-3'. The sense primer for amplification of the FN3 domain was