Activation by Adenosine Triphosphate in the Phosphorylation Kinetics of Sodium and Potassium Ion Transport Adenosine Triphosphatase

Adenosine 3':5'-monophosphate-regulated phosphoprotein system of neuronal membranes. I. Solubilization, purification, and some properties of an endogenous phosphoprotein.

Depolarization-induced phosphorylation of specific proteins, mediated by calcium ion influx, in rat brain synaptosomes.

Regulation of Endogenous Phosphorylation of Specific Proteins in Synaptic Membrane Fractions from Rat Brain by Adenosine 3':5'-Monophosphate

Regulation of protein kinase C by lysophospholipids. Potential role in signal transduction.

Publisher Summary This chapter discusses 32 P-labeling of a (Na + + K + )-ATPase intermediate, and describes the preparation of [γ- 32P ] ATP and phosphorylated intermediate. For the preparation of [γ- 32P ] ATP, the reaction is carried out at about 23° in the lead-glass bottle in which the 32 P comes. The reagents are added in the sequence given. Neutralization at step 2 is tested by adding a tiny drop to pH paper. Any pH between four and eight is satisfactory at this point. The cofactor solution (3) may be stored frozen. During the addition of the MgC1 2 (step 8), the reaction mixture is swirled gently in order to minimize the possibility of precipitating [ 32 P] MgNH 4 PO 4 . Formation of this precipitate delays labeling of ATP for about 10 hours. The enzyme preparations may provide enough P i and NH 4 + to form the precipitate if the procedure given is modified. For the preparation of phosphorylated intermediate, the reaction is carried out at 0° with rapid stirring. The reagents are added in the sequence given. Five seconds after the addition of the radioactive ATP, the reaction is stopped by the addition of 35 ml of 0.3 M HCI04 or trichloroacetic acid (fresh) containing 0.6 mM unlabeled ATP. The acid is at 0°, and the ATP is added to it less than 1 hour before the reaction is carried out. Formation of the intermediate is poorly reversible by ADP so that greater efficiency of incorporation of radioactivity could probably be obtained if the amount of carrier ATP were reduced.

R.L. Post

Papers

[118] 32P-labeling of a (Na+ + K+)-ATPase intermediate