A microassay for the determination of soluble and membrane-bound glutamate decarboxylase activity--influences of cations, lipid composition, and pyridoxal 5'-phosphate on the glutamate decarboxylase binding to liposomes.
A radiochemical microassay for soluble and membrane-bound glutamate decarboxylase (GAD) is described. Up to 180 samples can be determined per day with a variation coefficient of 2%. The method detects newly synthesized gamma-amino-n-butyric acid in the picomole range and can easily be applied to other enzymes whose substrate and product differ by charge. In an aqueous homogenate of brain (1 + 10; w/v) about 15% of the total GAD activity are spun down by centrifugation (1 h, 100,000g) increasing to 35% of the total GAD activity in solutions with 8 mM calcium chloride or 100 mM potassium acetate. There is similar dependence on the cation concentration when GAD binds to phospholipid vesicles (liposomes) as well as dependence on lipid concentration and lipid composition. The coenzyme pyridoxal 5'-phosphate has no influence on GAD binding to liposomes.
Hagel
C
C
Department of Neurochemistry, Psychiatric University Clinic, Hamburg, Federal Republic of Germany.
Fleissner
A
A
Seifert
R
R
D000818:Animals; D002250:Carbon Radioisotopes; D005968:Glutamate Decarboxylase / Q000378:metabolism*; D005971:Glutamates / Q000378:metabolism; D018698:Glutamic Acid; D008081:Liposomes / Q000378:metabolism; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism; D008832:Microchemistry / Q000379:methods; D011485:Protein Binding; D051381:Rats; D012995:Solubility; D005680:gamma-Aminobutyric Acid / Q000096:biosynthesis
D002250:Carbon Radioisotopes; D005971:Glutamates; D008081:Liposomes; D008565:Membrane Proteins; D018698:Glutamic Acid; D005680:gamma-Aminobutyric Acid; D005968:Glutamate Decarboxylase