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We studied the properties of calcium-activated ATPases present in preparations of isolated presynaptic nerve ending (synaptosome) and its subfractions from mouse brain. ATPase activity in the preparation was stimulated by Ca2+ and by Mg2+, but not by Na+ and K+, when each was added alone. The substrate specificities were found to be similar. The ATPases hydrolyzed only the high-energy phosphate bond and similar activity was exhibited for all nucleoside triphosphates tested (ATP, CTP, GTP, UTP). Moreover, the enzymes were insensitive to mitochondrial markers and to ouabain, but were inhibited by La3+. La3+ produced uncompetitive inhibition of Ca2+-ATPase in intact synaptosomes. Inhibition by La3+ was greatly increased after lysis of the synaptosomes, suggesting that the active sites of the enzymes may be on the cytosolic face of the membranes. The Ca2+-ATPase activity in synaptosomes was increased by increasing concentrations of external K+, suggesting that Ca2+ influx may be involved The Ca2+-ATPase in synaptosomal plasma membranes and synaptic vesicles had higher specific activities than those of intact synaptosomes and were activated, both in the presence and the absence of Mg2+, by Ca2+ concentrations approximating the intracellular level (10(-7) M). It is concluded that the nonmitochondrial synaptosomal Ca2+-ATPase may play an important role in the regulation of intracellular Ca2+.

Braunstein and Vilenkina (1) reported the presence of an enzyme (or enzyme system) in various animal tissues which forms glycine from serine, threonine, and certain other a-amino-/~-hydroxy acids. They named this enzyme glycinogenase. Vilenkina (2) showed that the enzyme system yielded as much glycine from DL-threonine as from DL-serine, that it operated selectively on the L-amino acid isomers; and that allothreonine was split more readily than was threonine. The products of the breakdown of threonine were glycine and acetaldehyde (3). The enzyme preparations used by Braunstein and Vilenkina were slices, homogenates, and acetone powder extracts of various tissues of various species. The present available information on this enzyme is limited to preparations not purified further than the extract of acetone powder of tissues.The present investigation was undertaken to isolate a more purified enzyme preparation and to study its general chemical characteristics, substrate specificity, and kinetics. The cleavage of threonine into glycine and acetaldehyde is similar to the reverse of an aldol condensation and threonine is the chief substrate commonly present in nature, serine not being attacked. Consequently, "threonine aldolase" appears to be a more appropriate name, and the enzyme is so designated in this paper. Experimental MethodsMeasurement of Threonine Aldolase Activity.--Threonine aldolase activity was estimated by measuring the rate of acetaldehyde formation. Incubations were carried out in tightly stoppered culture tubes, or in stoppered 10 ml. flasks, for 30 minutes at 37.3°C. The enzyme assay system contained, unless otherwise stated, 2 ml. of 0.15 ~t phosphate buffer, pH 7.6-7.7; 0.25 ml. of 0.25 M DL-allothreonine (or other amino acid) and 1 ml. of enzyme solution. When other components had to be added, the incubation volume was maintained at a total of 3.25 ml. by reducing the amount of phosphate and adding water when necessary. After incubation, the tubes were immediately * Aided by research grants from the Hobson Memorial

The properties of Ca2+-activated and Mg2+-activated ATPases of nerve endings from mouse brain were investigated. Ca2+ and Mg2+ each can activate ATP hydrolysis in synaptosomes and its subfractions. Both Ca2+-ATPase and Mg2+-ATPase exhibit high and low affinity for their respective cations. At millimolar concentrations of Ca2+ or Mg2+, several nucleoside triphosphates could serve as substrate for the two enzymes and their specific activities were about three to four times higher in synaptic vesicles than in synaptosomal plasma membranes (SPM). Both in SPM and in synaptic vesicles the relative activity in the presence of Ca2+ was in the order of CTP greater than UTP greater than GTP = ATP, but with Mg2+ the activity was higher with ATP than with the other three triphosphates. Mg2+-ATPase was more active than Ca2+-ATPase in SPM, but in synaptic vesicles the two enzymes exhibited similar activity. Kinetic studies revealed that Mg2+-ATPase was inhibited by excess ATP and not by excess Mg2+. The simultaneous presence of Na+ + K+ stimulated Mg2+-ATPase and inhibited Ca2+-ATPase activity in intact synaptosomes and SPM. The stimulation of Mg2+-ATPase by Na+ + K+ was further increased by increasing Mg2+ concentration and was inhibited by Ca2+ and by ouabain. When Ca2+ and Mg2+ are present together in SPM or synaptic vesicles, the total Pi liberated by the two cations may either increase or decrease, depending on their relative concentrations. Kinetic analyses indicate that Ca2+ and Mg2+ bind independently to the enzyme alone or together at different sites. The results suggest that Ca2+-ATPase and Mg2+-ATPase in SPM or synaptic vesicles may be separate and distinct systems.

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