KINETICS AND SUBCELLULAR-LOCALIZATION OF SPECIFIC [PHORBOL-H-3 12,13-DIBUTYRATE BINDING BY MOUSE-BRAIN

Abstract

The specific binding of [3H]phorbol 12,13-dibutyrate ([3H]PDBU) [a tumor promoter] to particulate preparations from mouse brain was further characterized. Kinetic analysis, using a filtration assay to measure binding, yielded a 2nd-order rate constant at 23.degree. C of 3.75 .times. 107 M-1 min-1 and a 1st-order dissociation rate constant of 0.21 min-1. The Kd of 5.6 nM calculated from the kinetic data agreed well with the value determined previously in equilibrium binding studies. The Kd 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 (.DELTA.H.degree. = +0.4 kcal/mol) and a large increase in entropy (.DELTA.S.degree. = +38 entropy units). Such values are characteristic for hydrophobic interactions. The dissociation rate constant for binding, in contrast to the Kd, varied dramatically with temperature. The half-time for release ranged from 1.75 min at 30.degree. C to 62 min at 4.degree. C. The Kd for binding was Ca2+ sensitive; chelation of Ca2+ by ethylene glycol bis(.beta.-aminoethyl ether) N,N''-tetraacetic acid increased the Kd 2.4-fold. Upon subcellular fractionation, the specific [3H]PDBU binding activity was exclusively particulate; no binding to cytosol was detectable. Binding did not correlate with nuclear or mitochondrial markers. A broader distribution of binding activity was seen on sucrose density gradients than for Na+-K+-adenosine triphosphatase activity or binding of quinuclidinyl benzilate (a muscarinic cholinergic antagonist). The location of specific [3H]PDBU binding to the plasma membrane therefore remains uncertain.