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Glutamine Synthetase and Glutamine Synthetase‐Like Protein from Human Brain
Author(s) -
Boksha Irina S.,
Tereshkina Elena B.,
Burbaeva Gulnur Sh.
Publication year - 2000
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1046/j.1471-4159.2000.0752574.x
Subject(s) - glutamine synthetase , hydroxylamine , glutamine , enzyme , biochemistry , transferase , microbiology and biotechnology , protein subunit , antiserum , biology , gel electrophoresis , chemistry , amino acid , antibody , gene , immunology
Glutamine synthetase (GS; EC 6.3.1.2), a key enzyme ofglutamate metabolism, and another enzyme possessing highhydroxylamine‐L‐glutamine transferase activity comparable to that of GS andtermed GS‐like protein (GSLP) were purified from human brain concurrently. Intwo‐dimensional electrophoresis, GS subunits migrate to at least six differentpositions (44 ± 1 kDa, pl = 6.4‐6.7), whereas GSLP subunits migrate toat least four different positions (54 ± 1 kDa, pl = 5.9‐6.2).Dependences of enzymatic activity in the transferase reaction onconcentrations of Mn 2+ and Mg 2+ for GS and GSLP aredifferent. High immunological cross‐reactivity between GS and GSLP wasobserved in ELISA. Nevertheless, antisera were raised to GS and GSLP, and amethod was developed for the separate detection of GS and GSLP in brainextracts by enzyme‐chemiluminescent amplified (ECL) immunoblotting. Thedistribution of GS and GSLP immunoreactivities between soluble protein andcrude mitochondrial fractions indicates tighter association with theparticulate fraction for GSLP than for GS. The results from activitymeasurements suggest that the hydroxylamine‐L‐glutamine transferase activitymeasured routinely in protein extracts from brain is the sum of GS and GSLPactivities. Similarly, immunoreactivity evaluated by ELISA is a sum ofimmunoreactivities of GS and GSLP. The relative contributions of GS and GSLPto the total immunoreactivity can be evaluated by ECL‐immunoblotting.