Isolation, purification and partial characterization of tropomyosin and troponin subunits from the lobster tail muscle.
In a search for an invertebrate muscle from which the muscle regulatory proteins could be obtained in a great quantity and at high homogeneity, the regulatory proteins, tropomyosin (Tm) and three subunits of troponin (Tn), have been isolated from the lobster tail muscle, purified and partially characterized. The calcium-sensitive ATPase of lobster myofibril was restored when purified lobster Tm and lobster Tn were added to actin. Quantitative SDS-polyacrylamide gel electrophoresis showed that the lobster muscle contains actin, Tm, Tn with a molar ratio 7:1:1 and that lobster Tn consists of three subunits, one of each I, C and T. Each subunit was identified according to its effect on the acto-S1 ATPase rate. The isomer composition in each fraction of purified Tn subunit and in Tm are different from the rabbit skeletal muscle proteins; Tm consists of a single species of polypeptide of M(r) 38,000; the TnT fraction appears to be homogeneous with M(r) 43,000; the TnI fraction contains five isomers, all showing similar isoelectric pH, differing in M(r) in the range from 28,000 to 31,000; two TnC fractions contain three isomers in total with a range of M(r) from 18,500 to 19,000. Further study of the lobster Tm elucidated that digestion by carboxypeptidase A gave rise to a homogeneous preparation of truncated and non-polymerizable Tm which is devoid of 11 residues at the C-terminus of the molecule. The C-terminal amino acid sequence of 11 residues is homologous to the thoracic isomer generated from Drosophila melanogaster Tm-I gene. The present study indicated that, despite heterogeneities owing to the occurrence of isomers, the lobster regulatory proteins serve as an invertebrate source of the proteins for structural and biophysical studies, alternative to vertebrate counterparts.
Miegel
A
A
European Molecular Biology Laboratory at DESY, Hamburg, Germany.
Kobayashi
T
T
Maéda
Y
Y
D000205:Actomyosin / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002852:Chromatography, Ion Exchange; D004331:Drosophila melanogaster / Q000378:metabolism; D007526:Isoelectric Point; D007536:Isomerism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D008121:Nephropidae / Q000737:chemistry*; D011817:Rabbits / Q000378:metabolism; D014335:Tropomyosin / Q000302:isolation & purification*; D014336:Troponin / Q000302:isolation & purification*
D014335:Tropomyosin; D014336:Troponin; D000205:Actomyosin; D002118:Calcium