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Brain Glutamate Decarboxylase: Properties of Its Calcium‐Dependent Binding to Liposomes and Kinetics of the Bound and the Free Enzyme
Author(s) -
Covarrubias Manuel,
Tapia Ricardo
Publication year - 1980
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.1471-4159.1980.tb11261.x
Subject(s) - glutamate decarboxylase , liposome , chemistry , egta , pyridoxal phosphate , biochemistry , phosphatidylserine , enzyme , phosphatidylethanolamine , phosphatidylcholine , phospholipid , calcium , cofactor , organic chemistry , membrane
In the present work we describe several properties of the Ca 2+ ‐dependent binding of glutamate decarboxylase (GAD) to phosphatidylcholine‐phosphatidylserine liposomes. The binding occurs very rapidly, is dependent on temperature in the range 23–37°C, is inhibited up to 35% by K + in a concentration‐dependent manner and is slightly increased when the dielectric constant of the medium is decreased by 3% ethanol. The association of GAD and liposomes is very firm, since EGTA displaces only 40% of the bound enzyme and Triton X‐100 about 55%. Since apparently only part of the total GAD is able to bind to the liposomes and in a previous study two forms of GAD activity have been identified kinetically, we compared the activations by pyridoxal 5′‐phosphate (PLP) of the soluble and the bound GAD, as well as their inhibition by PLP oxime‐ O ‐acetic acid. The bound GAD was activated 150–265% by 10 −6 to 10 −4 m ‐PLP, whereas the activation of GAD that remained soluble was only 65–110% in the same PLP concentration range. In the absence of PLP, the bound GAD was less inhibited by the PLP oxime‐ O ‐acetic acid than the soluble GAD, but the inhibition was similar when 0.1 m m ‐PLP was added. In contrast, activity of both the soluble and the bound GAD was totally blocked by aminooxyacetic acid. Endogenous PLP did not bind to liposomes under the experimental conditions inducing GAD binding. We conclude that the binding of GAD to negatively charged liposomes is primarily ionic. Furthermore, the GAD molecules that bind to the liposomes seem to be deficient in free PLP and therefore, are probably more susceptible to regulation by the coenzyme. These conclusions may be relevant to the hypothesis of a coupling between synthesis and release of GABA in inhibitory nerve endings.