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Kinetics and Subcellular Localization of Specific [³H]Phorbol 12,13-Dibutyrate Binding by Mouse Brain¹

Department of Pharmacology, Harvard Medical School, Boston, Massachusetts 02115

The specific binding of [³H]phorbol 12,13-dibutyrate ([³H]-PDBU) to particulate preparations from mouse brain has been further characterized. Kinetic analysis, using a filtration assay to measure binding, yielded a second-order rate constant at 23° of 3.75 x 10⁷ M^–1 min^–1 and a first-order dissociation rate constant of 0.21 min^–1. The K_d of 5.6 nM calculated from the kinetic data agreed well with the value determined previously in equilibrium binding studies. The K_d for [³H]PDBU binding varied only slightly with temperature. From its temperature dependence, [³H]PDBU binding appeared to be associated with a small increase in enthalpy (H° = +0.4 kcal/mol) and a large increase in entropy (S° = +38 e.u.). Such values are characteristic for hydrophobic interactions. The dissociation rate constant for binding, in contrast to the K_d, varied dramatically with temperature. The half-time for release ranged from 1.75 min at 30° to 62 min at 4°. The K_d for binding was Ca²⁺ sensitive; chelation of Ca²⁺ by ethyleneglycolbis(ß-aminoethyl ether)N,N'-tetraacetic acid increased the K_d 2.4-fold. Upon subcellular fractionation, the specific [³H]PDBU binding activity was exclusively particulate; no binding to cytosol was detectable. Binding clearly did not correlate with nuclear or mitochondrial markers. On the other hand, a broader distribution of binding activity was seen on sucrose density gradients than for either Na⁺-K⁺-adenosine triphosphatase activity or binding of quinuclidinyl benzilate (a muscarinic cholinergic antagonist). The localization of specific [³H]PDBU binding to the plasma membrane therefore remains uncertain.

¹ Supported by Grant CA 22895 from NIH.

² Present address: Department of Biochemistry, Stanford Medical School, Palo Alto, Calif. 94305.

³ Exchange scientist under the United States-France Cancer Program, sponsored by the National Cancer Institute and Institut National de la Santé et de la Recherche Médicale. Present address: Centre National de la Recherche Scientifique, 7, Rue Guy-Mocquet, 94800 Villejuif, France.

⁴ Recipient of a Research Career Development Award from NIH. To whom requests for reprints should be addressed.

Received 11/17/80. Accepted 3/25/81.

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