This chapter discusses an in vitro method for back-titration of brain phosphoproteins to determine the consequences of behavioral treatments on the available phosphorylatable sites of such proteins. This back-titration technique, in the endogenous phosphorylation system, measures the net phosphate incorporated as a consequence of both kinase and phosphatase activity. The back-titration method is influenced by procedural factors that alter, from the time before sacrifice to the time of assay, the amount of phosphate covalently bound to substrate proteins. The influence of method of sacrifice, buffer system, subcellular fraction and gel conditions on the accuracy of back-titration thus requires detailed consideration. With the use of conventional biochemical techniques, the establishment of evidence indicating that different laboratories are working with the same phosphoproteins, the use of back-titration to establish the consequences of functioned behavioral manipulations, and the identification of a behaviorally responsive phosphoprotein as pyruvate dehydrogenase, the study of the macromolecular basis of behavior can be said to be established on a firm foundation. Understanding the function of brain phosphoproteins will require the identification of the sequence of cellular and molecular events set into motion by specific environmental conditions, particularly those involving learning and memory.
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