The Purification and Properties of a Nucleotide Pyrophosphatase of Rat Liver Nuclei

Abstract

cleotide pyrophosphatase activit’y was routinely assayed with UDP-glucose as the substrate. The test system (total volume, 3.00 ml) was 100 mM in Tris-HCI buffer (pH S.5), 5.2 mM in MgSO+ 0.27 mM in NADP, and 0.20 mM in UDP-glucose and contained nucleotide pyrophosphatase and 0.1 ml or more of a solution of phosphoglucomutase (0.09 mg per ml) and glucose 6-phosphate dehydrogenase (0.032 mg per ml) that was 70 mM in imidazole and 2 mM in MgS04. A control was prepared that contained the ingredients in the reaction system with the exception of UDP-glucose. When purified nucleotide pyrophosphatase was available, the pyrophosphatase, instead of the UDP-glucose, was sometimes left out of the control. The reaction was followed by measuring NADP reduction spectrophotometrically at 340 rnp at room temperature. When glucose l-phosphate (at 0.20 mM) was tested in the reaction system in place of UDP-glucose, the initial velocity was at least 0.34 pmole of NADPH formed per minute. This is at least 10 to 50 times the velocity obtained with UDP-glucose. One unit of nucleotide pyrophosphatase activity is defined as the amount of enzyme that catalyzes the formation of NADPH at an initial velocity of 1.00 pmole per minute. Activity is expressed as units per ml of enzyme, and specific activity is expressed as units per mg of protein. This assay was used with all substrates that could form glucose l-phosphate upon hydrolysis. The initial velocity varied linearly with the amount of enzyme added as seen in Fig. 1. Assay Method B for Nucleotide Pyrophosphatase Activity-As an alternative method the following procedure was used. The test system (total volume, 3.00 ml) was 100 mM in Tris-HCl buffer (pH 8.5) and 5.2 mM in MgS04, and contained nucleotide pyrophosphatase and the appropriate substrate. Controls were prepared by omission of enzyme or substrate. After addition of the substrate, the solutions were mixed and incubated at 37”. The reaction was followed by measuring loss of substrate, as described in the section “Other Measurements” below. When NAD was the substrate, nicotinamide was frequently included at 100 mM to inhibit any NAD nucleotidase activity. The specific activity was expressed as the micromoles of substrate destroyed per minute per mg of protein. When the substrate was ADP, all tubes were incubated at 37” for the