Summary
A new, extremely sensitive, solid phase radioimmunoassay has been developed to quantify tropomyosin levels in heart tissue homogenates. Specific antibody was coupled to Sepharose 4B and saturation levels of [125I]tropomyosin bound. Release of radiolabel into the supernatant portion occurred when heart homogenates, authentic tropomyosin or tropomyosin in the presence of homogenate were added to these immunobeads. Quantification of the amount of tropomyosin was based on the level of release effected by standard tropomyosin with and without homogenate. The assay was determined to be highly specific and sensitive for tropomyosin. Picomolar quantities of the protein were readily detectable. Linearity extended well over the range of 5–200 ng tropomyosin in the homogenate. The method can be applied to other proteins for quantification during embryonic development.
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References
BAILEY, K. (1948) Techniques in protein chemistry.Biochem. J. 43, 271–9.
CHAFOULEAS, J. G., DEDMAN, J. R., MUNGALL, R. P. & MEANS, A. R. (1979) Calmodulin development and application of a sensitive radioimmunoassay.J. biol. Chem. 254, 10262–7.
COHEN, C., CASPER, D. L. D., JOHNSON, J. P., NAUSS, K., MARGOSSIAN, S. S. & PARRY, D. A. D. (1972) Tropomyosin troponin assembly. InThe Mechanism of Muscle Contraction (edited by GORDON, J., LEWIS, M. and ALSTON, K.), Vol. 37, pp. 287–289. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory.
CUMMINS, P. & PERRY, S. V. (1973) The subunits and biological activity of polymorphic forms of tropomyosin.Biochem. J. 133, 765–77.
CUMMINS, P. & PERRY, S. V. (1974) Chemical and immunochemical characteristics of tropomyosins from striated and smooth muscle.Biochem. J. 141, 43–9.
DHOOT, G. K. & PERRY, S. V. (1979) Distribution of polymorphic forms of troponin components and tropomyosin in skeletal muscle.Nature 278, 714–8.
EISENBERG, E. & KIELLEY, W. W. (1974) Troponin-tropomyosin complex column chromatographic separation and activity of the three active troponin components with and without tropomyosin present.J. biol. Chem. 249, 4742–8.
FAIRBANKS, G., STECK, T. L. & WALLACH, D. F. H. (1971) Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane.Biochemistry 10, 2606–17.
GREENWOOD, F. C., HUNTER, W. M. & GLOVER, J. S. (1963) The preparation of131I-labelled human growth hormone of high specific radioactivity.Biochem. J. 89, 114–23.
HALES, C. N. & RANDLE, P. G. (1963) Immunoassay of insulin antibody precipitate.Biochem. J. 88, 137–46.
HOLBERTON, E. J. & GOLDSPINK, G. (1973) A radioimmunoassay for myosin in cultured skeletal muscle cells.Expl Cell Res. 79, 471–4.
KOHN, J. & WILCHEK, M. (1978) A colorimetric method for monitoring activation of sepharose.Biochem. biophys. Res. Commun. 84, 7–14.
LAEMMLI, U. K. (1970) Cleavage of structural proteins during assembly of the head of bacteriophage-T4.Nature 227, 680–5.
LEMANSKI, L. F., JOSEPH, X. & IYENGAR, M. K. (1975) Quantitation by radioimmunoassay of absolute amounts of myosin in embryonic hearts of cardiac lethal mutant axolotls (Ambystoma mexicanum).J. Cell Biol. 67, 239a.
LOWRY, D. H., ROSEBROUGH, N. J., FARR, A. L. & RANDALL, R. J. (1951) Protein measurement with the folin phenol reagent.J. biol. Chem. 193, 265–75.
MAK, A. S., LEWIS, W. G., & SMILLIE, L. B. (1979) Amino acid sequences of rabbit skeletal β- and cardiac tropomyosins.FEBS Lett. 105, 232–4.
MAK, A. S., SMILLIE, L. B. & STEWART, G. R. (1980) A comparison of the amino acid sequences of rabbit skeletal muscle α- and β-tropomyosins.J. biol. Chem. 255, 3647–51.
MARCHALONIS, J. J. & WELTMAN, J. K. (1971) Relatedness among proteins: a new method of estimation and its application to immunoglobulins.Comp. Biochem. Physiol. 38, 609–25.
NISHIKAWA, A. H. & BAILON, P. (1975) Affinity purification methods improved procedures for cyanogen bromide reaction on agarose.Analyt. Biochem. 64, 268–75.
OBINATA, T., HAYASHI, I. & FISCHMAN, D. A. (1974) Direct isolation of native thin filaments from embryonic muscle cells.Devl Growth Diff. 16, 105–21.
POTTER, J. D. & HERRMANN, H. (1970) Studies of muscle development identification of tropomyosin and troponin and quantitation of tropomyosin in embryonic and mature chick leg muscle.Archs Biochem. Biophys. 141, 271–7.
ROY, R. K., MABUSHI, K., SARKAR, S., MIS, C. & STRETER, F. A. (1979) Changes in tropomyosin subunit pattern in chronic electrically stimulated rabbit fast muscles.Biochem. biophys. Res. Commun. 89, 181–7.
SODEK, J., HODGES, R. S. & SMILLIE, L. B. (1978) Amino acid sequence of the rabbit skeletal muscle α-tropomyosin, the COOH-terminal half (residues 142 to 284).J. biol. Chem. 253, 1129–36.
SPOONER, B. S., MORGAN, J. L., HOLLADAY, C. K. & BRIGHT, G. R. (1979) Structural differences in actins revealed by radioimmunoassay.J. Cell Biol. 83, 325a.
STONE, D. & SMILLIE, L. B. (1978) The amino acid sequence of rabbit skeletal α-tropomyosin, the NH2-terminal half and complete sequence.J. biol. Chem. 253, 1137–44.
STORTI, R. V., COEN, D. M. & RICH, A. (1976) Tissue-specific forms of actin in developing chick.Cell 8, 521–7.
WEGNER, A. (1979) Equilibrium of the actin-tropomyosin interaction.J. molec. Biol. 131, 839–53.
WELTMAN, J. K. & DOWBEN, R. M. (1973) Relatedness among contractile and membrane proteins: evidence for evolution from common ancestral genes.Proc. natn. Acad. Sci. 70, 3230–4.
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Moore, P.B., Lemanski, L.F. A radioimmunoassay method for quantification of α-tropomyosin in heart homogenates. J Muscle Res Cell Motil 3, 145–160 (1982). https://doi.org/10.1007/BF00711939
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DOI: https://doi.org/10.1007/BF00711939