PURIFICATION AND CHARACTERIZATION OF TRIOKINASE FROM PORCINE KIDNEY

In order to be able to use triokinase for the enzymatic assay of tissu e glyceraldehyde, we purified the enzyme to homogeneity from porcine k idney and characterized its biochemical properties. The purification w as performed by polyethylene glycol fractionation, anion exchange chro matography, hydroxyapatite chromatography, hydrophobic chromatography, and gel filtration. The enzyme was purified 937-fold from the crude e xtract with an overall yield of 28 %. It had a molecular weight of 122 ,000 and was a dimer composed of identical subunits. The optimal pH an d optimal temperature were 7.0 and 60 degrees C, respectively. This en zyme was stable when incubated at pH 7.0 at 40 degrees C for 1 h in th e presence of 0.1 mg/ml bovine serum albumin, No loss of activity occu rred for at least 1 month when the enzyme was stored at 4 degrees C in the presence of 1 mM dithiothreitol and 15 mM NaN3 under N-2. Only th ree compounds, i.e., D-glyceraldehyde, dihydroxyacetone, and glycolald ehyde, acted as the substrate of the enzyme, having Km's of 11, <5, an d 260 mu M, respectively. The Km for ATP-Mg2+ was 68 mu M. These resul ts indicate that porcine kidney triokinase has properties advantageous for the glyceraldehyde assay using glyceraldehyde-3-phosphate dehydro genase as a coupling enzyme.