Purification and properties of glucose-1-phosphate uridylyltransferase fromNeurospora crassa

A method was developed to assay glucose-1-phosphate uridylyltransferase and 2-acetamido-2-deoxyglucose-1-phosphate uridylyltransferase by separation and quantitation of the corresponding sugar nucleotides by HPLC. Glucose-1-phosphate uridylyltransferase (GPUT) fromNeurospora crassa was purified by a method involving ion-exchange, gel filtration, adsorption, and affinity chromatographic procedures. The enzyme was stable until the last step of purification, after which it became extremely labile, apparently due to disaggregation. With the purified enzyme, kinetic properties of GPUT were determined. Polyacrylamide gel electrophoresis (PAGE) of the purified enzyme under nondenaturing conditions showed a single band which contained all the enzymatic activity. Denaturation of the enzyme with sodium dodecyl sulfate followed by PAGE resolved the single band into four polypeptides of different molecular masses. The minimal molecular mass of the enzyme was calculated to be 537,000 Da. This value was similar to that calculated by sucrose density sedimentation, 580,000 Da, but different from that estimated by gel filtration, 1,600,000 Da. It is proposed that the native enzyme is a trimer which may be disaggregated. By electron microscopy of negatively stained samples, the enzyme appeared in the form of rosettes 10 nm in diameter.