Location and partial purification of phosphate dependent glutaminase from rat small intestine.

Abstract

Phosphate dependent glutaminase (PDG) is a key enzyme in intestinal energy metabolism and hence has an impact on nutrient absorption and nutritional status of the whole animal. PDG is known to be a mitochondrial enzyme but its sub-mitochondrial location is still controversial. Due to its instability, PDG has never been purified from the small intestine. The sub-mitochondrial localization of PDG was investigated by tracking the release of PDG and that of marker enzymes for sub-mitochondrial compartments following fractionation of mitochondria using digitonin. The dependence of PDG activity on the membrane phospholipids was investigated using phospholipase A2 treatment while the orientation of the enzyme in mitochondria was probed using sulphydryl inhibitors Mersalyl (Mers) and N-ethylmaleimide (NEM). PDG was partially purified after solubilizing PDG using different methods including lyophilization combined with digitonin fractionation and sonication in the presence of a stabilizing buffer. Solubilized proteins were separated by gel filtration chromatography. SDS-PAGE and Western blotting were conducted on fractions collected to show protein profiles and location of PDG bands. Release of PDG was intermediate between that of cytochrome c oxidase and matrix enzymes. Phospholipase A₂ treatment exhibited a time dependent loss of PDG activity. In intact mitochondria Mers inhibited 97% and NEM inhibited 64% of PDG activity at 1 mM concentration; with a more pronounced effect when combined with sonication. Pre-incubation of mitochondria in a stabilizing buffer before solubilization activated PDG 37-fold. Partial purification was achieved after using Sephacryl S-300 HR. Coomassie stained SDS-PAGE confirmed the partial purification of PDG with bands on Western blot observed to be 63-65 kDa, 50 kDa and 42 kDa. In conclusion, the results suggest that intestinal PDG is localized in two sub-mitochondrial fractions with each displaying a different form: PDG with a molecular weight of 50 kDa being localized in the mitochondria matrix and a 63-65 kDa PDG being bound to the mitochondria inner membrane. The membrane bound PDG requires the presence of phospholipids to retain its activity.