D-Mannitol dehydrogenase from Absidia glauca. Purification, metabolic role, and subunit interactions.
When Absidia glauca was grown in minimal media with D-mannitol as the only source of carbon, an NAD+ specific D-mannitol dehydrogenase (EC 1.1.1.67) was induced. The crude extract also gave evidence of mannitol kinase, mannitol-1-phosphate dehydrogenase, phosphofructokinase, and L-iditol dehydrogenase activity. The heat labile purified preparation was judged enzymically homogeneous based on evidence derived from substrate specificity studies and activity staining, following disc gel electrophoresis. The enzymic monomer, with a weight of about 67000 daltons, slowly polymerizes when stored at -20 degrees C, giving a multiplicity of protein bands on electrophoresis distributed predominantly across a spectrum from dimer to pentamer, with enzymic activity resident predominantly in even multiples of the monomer. Depolymerization occurred rapidly (hours) when a frozen preparation was brought to and held between 4 and 20 degrees C. Aggregate fragmentation with sodium dodecyl sulfate showed a time-temperature dependence, terminating in a subunit component of 13000 daltons. pH optimum for polyol oxidation occurs at 9.6 (NaOH-glycine buffer) while ketose reduction proceeded most rapidly at pH 7.0-7.2 (phosphate buffer). A regulatory role is suggested for this enzyme based on dead-end inhibition by mannitol 1-phosphate, multiple enzyme forms, and its locus at the initiation site for mannitol utilization. The physiological relevance of low-temperature aggregation to regulatory control remains to be established.
D-Mannitol dehydrogenase from Absidia glauca. Purification, metabolic role, and subunit interactions. When Absidia glauca was grown in minimal media with D-mannitol as the only source of carbon, an NAD+ specific D-mannitol dehydrogenase (EC 1.1.1.67) was induced. The crude extract also gave evidence of mannitol kinase, mannitol-1-phosphate dehydrogenase, phosphofructokinase, and L-iditol dehydrogenase activity. The heat labile purified preparation was judged enzymically homogeneous based on evidence derived from substrate specificity studies and activity staining, following disc gel electrophoresis. The enzymic monomer, with a weight of about 67000 daltons, slowly polymerizes when stored at -20 degrees C, giving a multiplicity of protein bands on electrophoresis distributed predominantly across a spectrum from dimer to pentamer, with enzymic activity resident predominantly in even multiples of the monomer. Depolymerization occurred rapidly (hours) when a frozen preparation was brought to and held between 4 and 20 degrees C. Aggregate fragmentation with sodium dodecyl sulfate showed a time-temperature dependence, terminating in a subunit component of 13000 daltons. pH optimum for polyol oxidation occurs at 9.6 (NaOH-glycine buffer) while ketose reduction proceeded most rapidly at pH 7.0-7.2 (phosphate buffer). A regulatory role is suggested for this enzyme based on dead-end inhibition by mannitol 1-phosphate, multiple enzyme forms, and its locus at the initiation site for mannitol utilization. The physiological relevance of low-temperature aggregation to regulatory control remains to be established.