Purification and Characterization of Two Isoenzymes of DL-Glycerol-3-phosphatase from Saccharomyces cerevisiae

Abstract

The existence of specific DL-glycerol-3-phosphatase (EC 3.1.3.21) activity in extracts of Saccharomyces cer- evisiae was confirmed by examining strains lacking non- specific acid and alkaline phosphatase activities. Dur- ing purification of the glycerol-3-phosphatase, two isozymes having very similar molecular weights were isolated by gel filtration and anion exchange chroma- tography. By microsequencing of trypsin-generated peptides the corresponding genes were identified as previously sequenced open reading frames of unknown function. The two genes, GPP1 (YIL053W) and GPP2 (YER062C) encode proteins that show 95% amino acid identity and have molecular masses of 30.4 and 27.8 kDa, respectively. The intracellular concentration of Gpp2p increases in cells subjected to osmotic stress, while the production of Gpp1p is unaffected by changes of exter- nal osmolarity. Both isoforms have a high specificity for DL-glycerol-3-phosphate, pH optima at 6.5, and Km G3P in the range of 3–4 mM. The osmotic induction of Gpp2p is blocked in cells that are defective in the HOG-mitogen- activated protein kinase pathway, indicating that GPP2 is a target gene for this osmosensing signal transduction pathway. Together with DOG1 and DOG2, encoding two highly homologous enzymes that dephosphorylate 2-deoxyglucose-6-phosphate, GPP1 and GPP2 consti- tute a new family of genes for low molecular weight phosphatases.