Synaptosomes and synaptosome membrane vesicles from the brain of Mamestra configurata: Application to voltage-dependent and ATP-dependent Ca 2+ ion transport studies

Abstract

High yields of relatively pure, morphologically well-preserved, functionally competent synaptosomes were prepared from brains of moths of Mamestra configurata using a modified microscale Ficoll flotation technique. Typical preparations yielded 10 mg of synaptosomal protein per gram of moth brains. The moth brain synaptosomes were virtually free of endoplasmic reticulum and mitochondrial contaminants as judged from marker enzyme studies and electron microscopy. Voltage-dependent Ca 2+ ion transport was studied using the moth brain synaptosome preparations. Synaptosomes took up radioactive 45Ca 2+ from the incubation medium. The rate of uptake was increased up to three-fold when the synaptosomes were incubated in a depolarizing, high [K +] medium. Time course studies indicated that voltage-dependent Ca 2+ uptake was composed of an early (<2 sec) fast phase and a late (>10 sec) slow phase. ATP-dependent Ca 2+ ion transport was studied in moth brain synaptosome membrane vesicles prepared from synaptosomes by osmotic shock and purified on a second Ficoll gradient. The inside-out synaptosome membrane vesicles contained an ATP-dependent calcium ion pump which transported 45Ca 2+ from the incuation medium into the interior of the vesicle in the presence of ATP. The calcium ionophore A23187 rapidly released accumulated 45Ca 2+ from the vesicles. The maximal rate of ATP-dependent Ca 2+ transport occurred at a [Ca 2+ free] of 0.1 to 0.2 nM, indicating that the transport process has a very high affinity for Ca 2+ ions.