Purification and immunocharacterization of human brain glutamine synthetase and its detection in cerebrospinal fluid and serum by a sandwich enzyme immunoassay.
A modified procedure employing a single chromatographic step to purify glutamine synthetase from human brain is described. The enzyme was characterized by native, denaturing, and two-dimensional gel electrophoresis and immunoblotting. Yield and purity were assessed by enzyme activity assay and a newly developed sandwich enzyme immunoassay using a mouse monoclonal antibody against native sheep brain glutamine synthetase. The immunoassay detected glutamine synthetase protein in samples where the enzyme had been inactivated by repeated cycles of freezing and thawing, and in serum and cerebrospinal fluid where glutamine synthetase was undetectable by the enzyme activity assay. Native glutamine synthetase from human brain occurred as an octamer with an estimated molecular weight of 360-400 kDa. Under reducing and denaturing conditions, the enzyme dissociated into monomeric subunits with an estimated molecular weight of 44 kDa. The monomers were recognized by the monoclonal antibody on immunoblots but not in the sandwich enzyme immunoassay, suggesting that the antigenic site occurs once on each subunit. Both human and sheep brain glutamine synthetases were composed of three and four different types of subunits with isoelectric points ranging from 7.0-7.2 and 6.8-7.0, respectively.
Tumani
H
H
Specialty Laboratories, Inc., Santa Monica, CA 90404-3900, USA.
Shen
G Q
GQ
Peter
J B
JB
D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000201:enzymology*; D004789:Enzyme Activation; D005974:Glutamate-Ammonia Ligase / Q000097:blood* / Q000134:cerebrospinal fluid* / Q000302:isolation & purification; D006801:Humans; D007124:Immunoenzyme Techniques; D011233:Precipitin Tests; D011489:Protein Denaturation; D012756:Sheep
D005974:Glutamate-Ammonia Ligase