Microtubular proteins of Chlamydomonas reinhardtii. An immunochemical study based on the use of an antibody specific for the beta-tubulin subunit

Abstract

An immunochemical assay for tubulin subunits is described. The method is applied directly to homogenates of Chlamydomonas reinhardtii solubilized in sodium dodecyl sulfate (Na dodecyl-SO4), and it makes use of a two-dimensional electrophoresis system; the first separation is carried out by Na dodecyl-SO4-polyacrylamide gel electrophoresis and the second by electrophoresis into an agarose gel containing antibodies. Tubulin is precipitated in the form of a "rocket" and the method is made quantitative through the use of cells labeled with [35S]sulfate. The antiserum used in this assay was prepared in rabbits using beta subunit of tubulin purified from Chlamydomonas flagella by two preparative Na dodecyl-SO4-polyacrylamide gel electrophoreses. This antiserum and an antiserum to alpha subunit of tubulin from porcine brain, prepared for comparative study, were extensively characterized. Both antisera show specificity for the polypeptide used as antigen and react with the native dimeric tubulin. The antiserum to beta subunit from Chlamydomonas flagella also forms immunoprecipitates with native brain tubulin and its beta subunit when used at high titer. In contrast, the antiserum to alpha subunit from porcine brain does not cross-react with Chlamydomonas tubulin. The immunochemical assay was applied to Chlamydomonas cells synchronized by a 12-h light/dark cycle. In cells collected during the light period (late G1), after removal of flagella, the content of tubulin is estimated to be 0.3% of total protein. As cells enter the dark period there is a striking increase in tubulin content which reaches a maximum just before cell division.