Immunological quantitation and localization of tubulin in adult rat heart isolated myocytes.

Isolated myocytes were purified from adult rat heart. Identification and localization of microtubules and quantitation of tubulin in these cells were performed by immunochemical procedures. Antibodies were raised against brain tubulin and purified by affinity chromatography. An enzyme-linked immunosorbent assay, ELISA, was developed for quantitation of tubulin. It allowed the measurement of 10 to 500 ng of tubulin. Tubulin content in adult rat cardiac myocytes was found to be approximately 10 micrograms per 100 mg of the total protein content. By means of a double immunofluorescence technique, the microtubule network, identified with antitubulin, was studied in reference to the sarcomeric A band labeled with antibodies specific to myosin heavy chains. The basis for identifying the microtubule network have included the use of specific antitubulin immunoglobulins and the sensitivity of the specific labeling of the network to antimitotic drugs and low temperature. It was found that microtubules were organized mainly around the nuclei, with important concentrations at the poles, showing extensions in the cone and in the cytoplasm as loosely organized loops. The shape of adult cardiac myocyte was not dependent upon the integrity of the microtubule network.