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Isolation of Nerve Terminals from Crustacean Muscle
Author(s) -
Santiapillai Nirmala F.,
Gray Sally R.,
Phillips R. Elizabeth,
Richardson Peter J.
Publication year - 1989
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 - 10.1111/j.1471-4159.1989.tb08548.x
Subject(s) - glutamate receptor , glutamate decarboxylase , biochemistry , glutamatergic , population , biology , glutamate dehydrogenase , differential centrifugation , biophysics , chemistry , enzyme , receptor , demography , sociology
A method for the isolation of γ‐aminobutyric acid‐ergic (GABAergic) and glutamatergic terminals from crustacean muscle was developed, using differential centrifugation and sucrose density gradient centrifugation. Individual fractions were assessed using a variety of markers. One fraction was isolated which showed 40‐fold purification of glutamate decarboxylase with a yield of 12%. This fraction was enriched in GABA, glutamate, glutamate dehydrogenase, and 5′‐nu‐cleotidase, but not in NADPH cytochrome c reductase. This fraction possessed an uptake system for GABA and glutamate with apparent kinetic constants of K m = 50 μ M, V max = 250 pmol/min/mg of protein and K m ‐ 183 μ M, V max = 219 pmol/min/mg of protein, respectively. Electron microscopy showed nerve terminal profiles and a heterogeneous population of membrane vesicles. This fraction contained 3.4 nmol ATP/mg of protein which was stable for 30 min at 12°C, and was also able to synthesise ATP from exogenous adenosine. The terminals released labelled GABA and glutamate in a Ca 2+ ‐dependent fashion on depolarisation. No release of ATP was detected. It is concluded that viable nerve terminals have been isolated which could be used as model systems for the study of GABAergic and glutamatergic neuro‐chemistry.