Kinetic, ESR, and trapping evidence for in vivo binding of Mn(II) to glutamine synthetase in brain cells.
Mn(II) has been proposed as a potential modulator of various important CNS enzymes, particularly glutamine synthetase, which is compartmentalized in the cytoplasm of glia. Previous studies demonstrated that total glial Mn(II) was 50-75 microM, of which 30-40% occurs in the cytoplasm. In the present study, electron spin resonance (ESR) was used to determine that the concentration of free cytoplasmic Mn(II) in cultured chick glial cells is 0.8 (+/- 0.2) microM, very near Kd for the GS-Mn(II) complex. No free Mn(II) could be detected in glial mitochondria. Association of Mn(II) with brain glutamine synthetase (GS) was assessed under in vivo conditions in the presence of millimolar Mg(II) by trapping bound 54Mn(II) ions in the active site with irreversible inhibitors, namely methionine-sulfoximine (MSOX) or specific analogues thereof plus ATP. Ovine brain tissue was lysed directly into buffer containing Mn(II), 3 mM Mg(II), 1 mM MSOX, 1 mM ATP, 200 mM KCl, and 20 mM NaCl. Alternatively, primary cultures of chick glial cells were permeabilized into these inactivation mixtures. alpha-Methyl-D,L-prothionine-S,R-sulfoximine was used to specifically inhibit the mechanistically-related enzyme gamma-glutamyl-cysteine synthetase prior to specific inactivation of GS by alpha-ethyl-D,L-methionine-S,R-sulfoximine. Even in the presence of 2-3 mM Mg(II), with only 5-10 microM Mn(II) present, approximately 20-30% of GS subunits were trapped with bound Mn(II). These results indicate that brain GS exhibits a high degree of specificity for binding Mn(II) over Mg(II) and that Mn(II) binds to GS to a significant extent under in vivo conditions.
Wedler
F C
FC
Dept. of Molecular & Cell Biology, P.M. Althouse Laboratory, Pennsylvania State University, University Park 16802.
Ley
B W
BW
D000818:Animals; D001921:Brain / Q000166:cytology / Q000201:enzymology*; D002478:Cells, Cultured; D002642:Chick Embryo; D003600:Cytosol / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D005974:Glutamate-Ammonia Ligase / Q000378:metabolism*; D007700:Kinetics; D008345:Manganese / Q000378:metabolism*; D009457:Neuroglia / Q000378:metabolism
D008345:Manganese; D005974:Glutamate-Ammonia Ligase