A 63 KDA PHOSPHOPROTEIN UNDERGOING RAPID DEPHOSPHORYLATION DURING EXOCYTOSIS IN PARAMECIUM CELLS SHARES BIOCHEMICAL CHARACTERISTICS WITH PHOSPHOGLUCOMUTASE

We have enriched phosphoglucomutase (PGM; EC 5.4.2.2) similar to 20-fo ld from Paramecium tetraurelia cells by combined fractional precipitat ion with (NH4)(2)SO4, gel filtration and anion-exchange chromatography yielding two PGM peaks. Several parameters affecting PGM enzymic acti vity, molecular mass and pi were determined. Phosphorylation studies w ere done with isolated endogenous protein kinases. Like the 63 kDa pho sphoprotein PP63, which is dephosphorylated within 80 ms during synchr onous trichocyst exocytosis [Hohne-Zell, Knell, Riedel-Gras, Hofer and Plattner (1992) Biochem. J. 286, 843-849], PGM has a molecular mass o f 63 kDa and forms of identical pI. Since mammalian PGM activity depen ds on the presence of glucose 1,6-bisphosphate (Glc-1,6-P-2) (which is lost during anion-exchange chromatography), we analysed this aspect w ith Paramecium PGM. In this case PGM activity was shown not to be lost , due to p-nitrophenyl phosphate-detectable phosphatase(s) (which we h ave separated from PGM), but also due to loss of Glc-1,6-P-2. Like PGM from various vertebrate species, PGM activity from Paramecium can be fully re-established by addition of Glc-1,6-P-2 at 10 nM, and it is al so stimulated by bivalent cations and is insensitive to chelating or t hiol reagents. The PGM which we have isolated can be phosphorylated by endogenous cyclic-GMP-dependent protein kinase or by endogenous casei n kinase. This results in three phosphorylated bands of identical mole cular mass and pI values, as we have shown to occur with PP63 after ph osphorylation in vivo (forms with pI 6.05, 5.95, 5.85). In ELISA, anti bodies raised against PGM from rabbit skeletal muscle were reactive no t only with original PGM but also with PGM fractions from Paramecium. Therefore, PGM and PP63 seem to be identical with regard to widely dif ferent parameters, i.e. co-elution by chromatography, molecular mass, phosphorylation by the two protein kinases tested, pi values of isofor ms, and immune-binding. Recent claims that PP63 ('parafusin') would no t be identical with PGM specifically in Paramecium are critically eval uated. Since some glycolytic enzymes are discussed as being associated with the Ca2+-release channel in muscle sarcoplasmic reticulum, and s ince sub-plasmalemmal Ca2+ stores in Paramecium closely resemble sarco plasmic reticulum, a possible function of PP63/PGM in exocytosis regul ation is discussed, particularly since dephosphorylation strictly para llels exocytosis.