Angel I, Fleissner A, Seifert R
Department of Neurochemistry, Psychiatric University Clinic, D-2000 Hamburg 20, F.R.G.
Neurochem Int. 1983;5(6):697-712. doi: 10.1016/0197-0186(83)90095-5.
Purified synaptic vesicles were isolated from hog cerebral cortex by a rapid procedure consisting of homogenization of cerebral cortex slices in iso-osmotic sucrose, differential centrifugation and sucrose density-gradient centrifugation. The purity of the vesicles was evaluated both biochemically and morphologically. The vesicles contained high amounts of ?-aminobutyrate (GABA) and acetylcholine at specific concentrations of 390 nmol/mg protein and 7.2 nmol/mg protein respectively. Glutamate decarboxylase, the enzyme which catalyses GABA formation, binds to the synaptic vesicles in a calcium-dependent manner. The percentage of glutamate decarboxylase bound to the vesicles increases from about 5% without calcium, reaching a plateau of about 60% at 4 mM Ca(2+). Magnesium in concentrations 0.2-10 mM has no significant effect on glutamate decarboxylase binding. Also in phospholipid vesicles (small unilamellar phosphatidylserine-phosphatidylcholine. 2:1 liposomes) Ca(2+), but not Mg(2+), induced the binding of glutamate decarboxylase, reaching a plateau of 50% at 2 mM Ca(2+). Both in synaptic vesicles and in phospholipid vesicles the calcium-dependent glutamate decarboxylase binding seems to be specific, and not caused by unspecific association of proteins, since the specific binding (bound enzyme activity/mg bound protein) increases 3-fold from 0 to 4 mM Ca(2+). The functional role of this binding was studied in GAD containing vesicles by measuring the relationship between the accumulation of [(3)H]GABA, newly synthetized from [(3)H]glutamate, and the uptake of added [(14)C]GABA. No significant uptake of [(14)C]GABA was found under the experimental conditions used, whereas large amounts of [(3)H]GABA were found within the vesicles. It appears that the [(3)H]GABA accumulation process is functionally linked to [(3)H]GABA synthesis and is mediated by the membrane-bound glutamate decarboxylase. This synthesis-coupled uptake of GABA into synaptic vesicles possibly serves to bring about a plasticity effect in previously stimulated GABAergic nerve endings.
通过一种快速方法从猪大脑皮层中分离出纯化的突触小泡，该方法包括将大脑皮层切片在等渗蔗糖中匀浆、差速离心和蔗糖密度梯度离心。通过生化和形态学方法评估小泡的纯度。这些小泡分别含有高浓度的γ-氨基丁酸（GABA）和乙酰胆碱，其特定浓度分别为390 nmol/mg蛋白质和7.2 nmol/mg蛋白质。催化GABA形成的谷氨酸脱羧酶以钙依赖的方式与突触小泡结合。与小泡结合的谷氨酸脱羧酶的百分比在无钙时约为5%，在4 mM Ca(2+)时达到约60%的平台期。浓度为0.2 - 10 mM的镁对谷氨酸脱羧酶的结合没有显著影响。同样，在磷脂小泡（小单层磷脂酰丝氨酸 - 磷脂酰胆碱，2:1脂质体）中，Ca(2+)而非Mg(2+)诱导谷氨酸脱羧酶的结合，在2 mM Ca(2+)时达到50%的平台期。在突触小泡和磷脂小泡中，钙依赖的谷氨酸脱羧酶结合似乎都是特异性的，并非由蛋白质的非特异性结合引起，因为特异性结合（结合酶活性/mg结合蛋白）从0到4 mM Ca(2+)增加了3倍。通过测量从[(3)H]谷氨酸新合成的[(3)H]GABA的积累与添加的[(14)C]GABA的摄取之间的关系，研究了这种结合在含有谷氨酸脱羧酶的小泡中的功能作用。在所使用的实验条件下未发现[(14)C]GABA的显著摄取，而在小泡内发现了大量的[(3)H]GABA。似乎[(3)H]GABA的积累过程在功能上与[(3)H]GABA的合成相关，并由膜结合的谷氨酸脱羧酶介导。这种合成偶联的GABA摄取到突触小泡中可能有助于在先前受刺激的GABA能神经末梢产生可塑性效应。
