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Covalent interaction of [3H]‐dopamine with rat brain proteins in vivo and with the dopamine‐reuptake site of nerve endings in vitro

Isolated nerve endings have been demonstrated to undergo saturable, covalent interactions with [3H]‐dopamine under physiological conditions; and the reaction is greatly accelerated by flash photolysis with ultraviolet light. With intact nerve endings, under conditions where dopamine reuptake occurs, benztropine and cocaine (inhibitors of dopamine reuptake), but not atropine or haloperidol (a postsynaptic antagonist), prevent the reaction. The reaction also occurs in vivo following the intraventricular administration of [3H]‐dopamine, the reaction being greatest with mitochondria, followed by the nerve ending and myelin. With the use of sodium dodecylsulfate–gel electrophoresis, a number of proteins of varying molecular weight were labeled, and the pattern of labeling was similar in vitro and in vivo. One protein, with a MW of about 60,000 was labeled to an exceptionally high degree. A number of protein bands showed decreased radiolabeling in the presence of benztropine, a finding which suggests that they may be associated with the reuptake site. Both the addition of ascorbic acid and unlabeled dopamine inhibited the reactivity of [3H]‐dopamine, and the effects were concentration dependent. In the absence of photolysis, the reaction of [3H]‐dopamine to synaptic membranes attained saturation within 10 min, but with photolysis the reaction continued at a constant rate even after 20 min. The results are discussed in relation to the use of [3]‐dopamine as a photoaffinity label of the dopamine reuptake site and in relation to the nature of the reactions with and without photolysis.