Solubilization and Separation of Uridine Diphospho-d-glucose: β-(1 → 4) Glucan and Uridine Diphospho-d-glucose:β-(1 → 3) Glucan Glucosyltransferases from Coleoptiles of Avena sativa

摘要

The particulate glucan synthetase preparation isolated from a homogenate of oat coleoptiles at 4 C lost 65% of its original activity after 1 day when the UDP-d-glucose substrate concentration was 5 × 10⁻⁷m to 1.0 × 10⁻⁶m. Storage of the particulate enzyme at −20 C or in liquid nitrogen did not prevent the enzyme from losing its activity. Incorporation of 0.5% hovine serum albumin into the medium stabilized the particulate enzyme at 0 C for 6 days and for at least 2 weeks in liquid nitrogen.When the particulate enzyme was treated with 8% digitonin, 40 to 50% of its activity appeared in the 100,000g supernatant fraction. The particulate and digitonin-solubilized enzyme preparations synthesized both β-(1 → 4) and β-(1 → 3) glucosyl linkages from UDP-d-glucose, but β-(1 → 3) glucan was the main product at 1 × 10⁻³m UDP-d-glucose substrate. The activity of β-(1 → 4) glucan synthetase was stimulated at least 10-fold in the presence of MgCl2. A separation of β-(1 → 4) and β-(1 → 3) glucan synthetase activities could be achieved at 1 × 10⁻³m UDP-d-glucose when the digitonin-solubilized enzyme was adsorbed on a hydroxylapatite gel and then eluted with concentrated potassium phosphate buffer. The results indicate that the particulate enzyme contains two enzymes, one responsible for the synthesis of β-(1 → 4) and another β-(1 → 3) linkages in the glucan or glucans synthesized from UDP-d-glucose.