Adenosine triphosphate-dependent calcium-binding vesicles; magnesium, calcium adenosine triphosphatase and sodium, potassium adenosine triphosphatase: Distributions in dog brain

Abstract

Mg 2+,Ca 2+-ATPase, Na +,K +-ATPase, and ATP-dependent Ca 2+-binding were studied in dog brain. The significant characteristics of ATP-dependent Ca 2+-binding in a brain microsomal fraction include: (a) Maximum Ca 2+-binding (which corresponds to that of sarcoplasmic reticulum in the absence of oxalate): 120 nmoles/mg protein. Sr 2+ can also be accumulated by an energy-dependent process. (b) Rate: 55 nmoles Ca 2+/mg protein/min. (c) The release process follows slowly after maximum binding, but does not reach the original level at pH 7.4. (d) The vesicles which accumulate Ca 2+ may be mostly endoplasmic reticulum, because of their distribution at 0.8-0.6-0.4 m after sucrose density gradient centrifugation. Other minor portions, however, might arise from the cell membrane fraction. (e) Phospholipids are important constituents of the Ca 2+-accumulating system probably in a manner similar to the sarcoplasmic reticulum (SR). (f) It is different from the SR process, however, in that oxalate or phosphate has no effect on Ca 2+ uptake. (g) Deoxycholate appears to “damage” the ability to bind calcium. (h) Of the nucleotides tested, only ATP is effective. (i) Apparent optimum pH is 6.2. (j) Azide and ouabain are inactive; salyrgan has a biphasic effect causing an increase at low concentration and an inhibition at high concentrations. The noteworthy characteristics of the Mg 2+,Ca 2+-ATPase of the brain membrane preparation are (a) optimum Ca 2+ concentration is low, 30 μ m at 5 × 10 −3 m Mg 2+; (b) specific activity is about 18 μmoles P i/mg protein/hr and is more unstable than Na +,fK +-ATPase; (c) the activity appears to be latent, but is elicited by detergents; (d) it is probably located mainly in a membrane fraction similar to the Na +, K +-ATPase; (e) the K m for ATP is low (4.3 × 10 −5 M); (f) the optimum pH is 7.4; (g) of the nucleotides tested, only ATP is significantly hydrolyzed; (h) Sr 2+ and Ba 2+ can substitute for Ca 2+ to some extent; (i) phospholipids appear to be important constituents of this enzyme system; (j) salyrgan almost completely inhibits the activity while azide has no effect. A role of the Mg 2+,Ca 2+-ATPase in active extrusion of intracellular calcium is suggested. The Ca 2+-binding vesicles, possibly representing regions for endoplasmic reticulum, might control free intracellular calcium.