Molecular characterization of avian muscle titin.
Titin is an approximately 3000-kDa polypeptide that constitutes a set of elastic filaments that connect thick filaments to the Z-line in vertebrate striated muscle myofibrils. To characterize the primary structure of titin, three overlapping cDNA clones comprising 2.4 kilobases of avian muscle titin coding sequence were obtained from a cDNA library constructed from embryonic chick cardiac muscle RNA size-selected for large transcripts. Expression of one cDNA clone in Escherichia coli produced a fusion protein that reacted specifically with titin antibodies, and titin antiserum affinity-purified against this fusion protein reacted specifically with titin on immunoblots of chicken cardiac and skeletal muscle myofibrils. Indirect immunofluorescence localization with the fusion protein-specific antibodies demonstrated that the cDNA sequence was from the region of titin located in the myofibrillar A-band adjacent to the A/I junction. Derived amino acid sequences demonstrated a repeating pattern of fibronectin type III and immunoglobulin C2 motifs, as shown previously for a portion of rabbit skeletal muscle titin located in the central region of the A-band and for other myofibrillar proteins that bind to myosin. Differences between the rabbit and chicken titin sequences included a unique, serine-rich region in one motif, which represents a potential phosphorylation site. This is the first report of sequence information for avian titin from a previously uncharacterized portion of the titin molecule.
Tan
K O
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Department of Animal Science, Iowa State University, Ames 50011.
Sater
G R
GR
Myers
A M
AM
Robson
R M
RM
Huiatt
T W
TW
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D002642:Chick Embryo; D002645:Chickens; D003001:Cloning, Molecular; D064211:Connectin; D017124:Conserved Sequence; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli; D008565:Membrane Proteins / Q000096:biosynthesis; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D009206:Myocardium / Q000378:metabolism*; D011494:Protein Kinases; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D012091:Repetitive Sequences, Nucleic Acid; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid
D064211:Connectin; D018076:DNA, Complementary; D008565:Membrane Proteins; D009124:Muscle Proteins; D011994:Recombinant Proteins; D011494:Protein Kinases