Glutamine synthetase and glutamine synthetase-like protein from human brain: purification and comparative characterization.
Glutamine synthetase (GS; EC 6.3.1.2), a key enzyme of glutamate metabolism, and another enzyme possessing high hydroxylamine-L-glutamine transferase activity comparable to that of GS and termed GS-like protein (GSLP) were purified from human brain concurrently. In two-dimensional electrophoresis, GS subunits migrate to at least six different positions (44 +/- 1 kDa, pl = 6. 4-6.7), whereas GSLP subunits migrate to at least four different positions (54 +/- 1 kDa, pl = 5.9-6.2). Dependences of enzymatic activity in the transferase reaction on concentrations of Mn(2+) and Mg(2+) for GS and GSLP are different. High immunological cross-reactivity between GS and GSLP was observed in ELISA. Nevertheless, antisera were raised to GS and GSLP, and a method was developed for the separate detection of GS and GSLP in brain extracts by enzyme-chemiluminescent amplified (ECL) immunoblotting. The distribution of GS and GSLP immunoreactivities between soluble protein and crude mitochondrial fractions indicates tighter association with the particulate fraction for GSLP than for GS. The results from activity measurements suggest that the hydroxylamine-L-glutamine transferase activity measured routinely in protein extracts from brain is the sum of GS and GSLP activities. Similarly, immunoreactivity evaluated by ELISA is a sum of immunoreactivities of GS and GSLP. The relative contributions of GS and GSLP to the total immunoreactivity can be evaluated by ECL-immunoblotting.
Boksha
I S
IS
Laboratory of Neurochemistry, Mental Health Research Center of Russian Academy of Medical Sciences, Moscow, Russia.
Tereshkina
E B
EB
Burbaeva
G S
GS
D019732:Amide Synthases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D000818:Animals; D000918:Antibody Specificity; D001921:Brain / Q000201:enzymology*; D001923:Brain Chemistry; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005615:Freezing; D005974:Glutamate-Ammonia Ligase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D008928:Mitochondria / Q000737:chemistry / Q000378:metabolism; D009419:Nerve Tissue Proteins; D021122:Protein Subunits; D013347:Subcellular Fractions / Q000737:chemistry / Q000378:metabolism; D013552:Swine
D009419:Nerve Tissue Proteins; D021122:Protein Subunits; D019732:Amide Synthases; C417025:glutamine synthetase-like protein, human; D005974:Glutamate-Ammonia Ligase