Abstract
High affinity Ca2+-stimulated Mg2+-dependent ATPase activity of nerve ending particles (synaptosomes) from rat brain tissue appears to be associated primarily with isolated synaptic plasma membranes. The synaptic membrane (Ca2+ + Mg2+)-ATPase activity was found to exhibit strict dependence on Mg2+ for the presence of the activity, a high affinity for Ca2+ (K0.5 = 0.23 microM), and relatively high affinities for both Mg2+ and ATP (K0.5 = 6.0 microM for Mg2+ and KM = 18.9 microM for ATP). These kinetic constants were determined in incubation media that were buffered with the divalent cation chelator trans-cyclohexane-1,2-diamine-N,N,N',N'-tetraacetic acid. The enzyme activity was not inhibited by ouabain or oligomycin but was sensitive to low concentrations of vanadate. The microsomal membrane subfraction was the other brain subcellular fraction with a high affinity (Ca2+ + Mg2+)-ATPase activity which approximated that of the synaptic plasma membranes. The two membrane-related high affinity (Ca2+ + Mg2+)-ATPase activities could be distinguished on the basis of their differential sensitivity to vanadate at concentrations below 10 microM. Only the synaptic plasma membrane (Ca2+ + Mg2+)-ATPase was inhibited by 0.25-10 microM vanadate. The studies described here indicate the possible involvement of both the microsomal and the neuronal plasma membrane (Ca2+ + Mg2+)-ATPase in high affinity Ca2+ transport across membranes of brain neurons. In addition, they suggest a means by which the relative contributions of each transport system might be evaluated based on their differential sensitivity to inhibition by vanadate.
Highlights
Mga+-dependent The ATP-dependent transport of Ca2+has been viewed by ATPase activity of nerve ending particles (!vnaphmm)some investigators as a sequestration of Ca2+within vesicular fromrat braintissue appears tobe associatedprimarily structures present in nerve endings (5, 12, 13, 22, 23)
Tic membrane (Ca2+ Mga+)-ATPase activitywas transport in nerve terminal preparations(synaptosomal memfound to exhibit strict dependence on M g + for the branes) is a plasma membrane process that leads to Ca"
Enzymatic Marker Assays-The activity of the plasma membrane enzyme 5'-nucleotidase was measured as described by Edwards and Maguire (31)
Summary
Fractions-Whole brains were rapidly removed following cervical dislocation, minced, and homogenized in 20 volumes of cold sucrose-MgSO., medium in a Potter-Elvehjem glass homogenizer with a Teflon pestle (0.13 mm clearance) by eight strokes at 1000 rpm. Lyse In H20 fractions (4-9mg of protein/ml) were divided into smallaliquots, frozen rapidly in liquid Nz, and stored a t -80 "C until used. Calciumstimulated M$+-ATPase activity was initially tested in freshly isolated as well as rapidly frozen and thawed subcellular fractions. Enzymatic Marker Assays-The activity of the plasma membrane enzyme 5'-nucleotidase was measured as described by Edwards and Maguire (31).
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