Glycerol-3-phosphatase from Trypanosoma brucei brucei

Abstract

Glycerol-3-phosphatase activity was found in both bloodstream and procyclic form o f trypanosomes with a specific activity of 35 nmol/min/mg of protein and 28 nmol/min/mg of protein respectively. The localisation of the enzyme in bloodstream form of trypanosomes was identified as cytosolic using cell fractionation techniques followed by the purification of the enzyme using ion exchange chromatography and size exclusion chromatography. The purified enzyme had a specific activity of 100 μmol/min/mg protein and had an apparent molecular weight of 32.8 kDa estimated by Tricine SDS-PAGE. Characterisation studies revealed that the enzyme had a molecular weight of 33 kDa determined using a gel filtration technique which was in close agreement with that estimated from SDS-PAGE. The pI of glycerol-3- phosphatase was estimated by chromatofocusing the enzyme using a pH gradient during elution and the enzyme eluted at a pH of 5.0, which was consistent with values reported for other cytosolic enzymes. The activity profile of the enzyme was determined as a function of H+ concentration and showed a sharp optimum pH at 7. Glycerol-3-phosphatase required Mg2 for activity and was inhibited SO4 2- as well as Ba2+, Pb2+ and Ca2+. The enzyme has a Km of 2.7 mM for D,L-glycerol-3-phosphate and 1.6 mM for L-glycerol-3-phosphate. Approximately equal activity was observed with D,L-glycerol-3-phosphate, glucose-6-phosphate and fructose-6-phosphate indicating that the enzyme was not specific for glycerol-3-phosphate.