A purification procedure for L-myo-

A purification procedure for L-myo-inositol-1-phosphate synthase (EC 5.5.1.4) from yeast is described. The method routinely produces enrichments of 500-fold with 20-40% yields. In addition, a procedure for obtaining highly specific and purified antibody against the protein is described. The molecular weight of the native enzyme as determined by gel filtration is approximately 240,000. A single subunit of approximately Mr = 62,000 is detected upon sodium dodecyl sulfate-gel electrophoresis of the purified enzyme. The purified antibody was used to assay crude extracts of wild type and inositol-requiring mutants for the presence of cross-reacting material. Mutant ino1-13 produces an inactive but fully cross-reacting protein of a molecular weight identical with the wild type enzyme subunit. Mutant ino1-16 produces low levels of a fully active enzyme which appears to be more susceptible to proteolytic degradation. Mutants representing other unlinked loci (ino2 and ino4) do not produce cross-reacting protein. Based on this analysis, the ino1 locus is identified as the structural gene for the enzyme. Furthermore, it is shown that the Mr = 62,000 subunit is largely absent from crude extracts prepared from wild type yeast grown in the presence of repressing concentrations of inositol.

A purification procedure for L-myo-inositol-1-phosphate synthase (EC 5.5.1.4) from yeast is described. The method routinely. Produces enrichments of 500-fold with 20-40% yields. In addition a procedure, for obtaining highly specific and purified. Antibody against the protein is described. The molecular weight of the native enzyme as determined by gel filtration is. Approximately, 240 000. A single subunit of approximately Mr = 62 000 is, detected upon sodium dodecyl sulfate-gel electrophoresis. Of the purified enzyme. The purified antibody was used to assay crude extracts of wild type and inositol-requiring mutants. For the presence of cross-reacting material. Mutant ino1-13 produces an inactive but fully cross-reacting protein of a molecular. Weight identical with the wild type enzyme subunit. Mutant ino1-16 produces low levels of a fully active enzyme which appears. To be more susceptible to proteolytic degradation. Mutants representing other unlinked loci (ino2 and ino4) do not produce. Cross-reacting protein. Based on this analysis the INO1, locus is identified as the structural gene for the enzyme, Furthermore,. It is shown that the Mr = 62 000 subunit, is largely absent from crude extracts prepared from wild type yeast grown in the. Presence of repressing concentrations of inositol.