myo-Inositol-1-phosphate synthase. Characteristics of the enzyme and identification of its structural gene in yeast.
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.
D000906:Antibodies; D000936:Antigen-Antibody Complex; D002238:Carbohydrate Epimerases / Q000235:genetics*; D005796:Genes; D046911:Macromolecular Substances; D008970:Molecular Weight; D009154:Mutation; D007296:Myo-Inositol-1-Phosphate Synthase / Q000235:genetics* / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics
D000906:Antibodies; D000936:Antigen-Antibody Complex; D046911:Macromolecular Substances; D002238:Carbohydrate Epimerases; D007296:Myo-Inositol-1-Phosphate Synthase