The glycolytic enzyme enolase (EC 4.2.1.11) is active as dimers formed from three subunits encoded by different genes. The embryonic αα isoform remains distributed in many adult cell types, whereas a transition towards ββ and γγ isoforms occurs in striated muscle cells and neurons respectively. It is not understood why enolase exhibits tissue-specific isoforms with very close functional properties. We approached this problem by the purification of native ββ-enolase from mouse hindlimb muscles and by raising specific antibodies of high titre against this protein. These reagents have been useful in revealing a heterogeneity of the β-enolase subunit that changes with in i o and in itro maturation. A basic carboxypeptidase appears to be involved in generating an acidic β-enolase variant, and may regulate plasminogen binding by this subunit. We show for the

The yeast Tup1 and Ssn6 proteins form a transcriptional repression complex that represses transcription of a broad array of genes. It has been shown that the N-terminal domain of the Tup1 protein interacts with a region of the Ssn6 protein that consists of 10 tandem copies of a tetratricopeptide motif. In this work, we use a surface plasmon resonance assay to measure the affinity of the N-terminal domain of Tup1 for a minimal 3-TPR domain of Saccharomyces cerevisiae Ssn6 that is sufficient for binding to Tup1. This domain of Ssn6 binds with comparable affinity to S. cerevisiae and Candida albicans Tup1, but with 100-fold lower affinity to Tup1 protein containing a point mutation that gives rise to a defect in repression in vivo. Results from studies using analytical ultracentrifugation, CD spectroscopy, limited proteolysis, and 1 H NMR show that this domain of Tup1 is primarily ␣-helical and forms a stable tetramer that is highly nonglobular in shape. X-ray diffraction recorded from poorly ordered crystals of the Tup1 tetramerization domain contains fiber diffraction typical of a coiled coil. Our results are used to propose a model for the structure of the N-terminal domain of Tup1 and its interaction with the Ssn6 protein.

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