Pig liver glyceraldehyde-3-P dehydrogenase: Purification, crystallization, and characterization

Abstract

Glyceraldehyde 3-P dehydrogenase was purified approximately 250-fold from pig liver and crystallized. The purification procedure consisted of treating liver homogenates with zinc chloride, followed by ammonium sulfate fractionation and ion exchange chromatography. The enzyme was monodisperse in the ultracentrifuge with a sedimentation coefficient of s 20,w = 7.85 S. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed a single subunit band with an approximate molecular weight of 38,000. High-speed sedimentation equilibrium gave a molecular weight of 1.5 × 10 5. Incubation of the enzyme with ATP at 0 °C caused a loss of its dehydrogenase activity; some of the lost activity was regained upon warming to room temperature. Sucrose density gradient studies of the ATP-treated enzyme revealed a decrease in its sedimentation coefficient from 7.8 to 3.85 S. In the forward reaction direction, the K m for glyceraldehyde 3-P was 240 μ m and the K m for NAD was 12 μ m. In the backward reaction direction, the K m for NADH was 23 μ m and the K i for NAD was 850 μ m. Pig liver glyceraldehyde-3-P dehydrogenase resembles the rabbit muscle enzyme in that it apparently contains 2 to 3 mol of tightly bound NAD. However, it differs strongly from that enzyme in its rate and extent of inactivation by ATP at 0 °C and by urea; the pig liver enzyme, like the yeast enzyme, dissociates much more slowly and much less completely than the rabbit muscle enzyme under comparable conditions.