Catecholamine Binding Macromolecule in Soluble Fraction of Rat Brain

Abstract

A macromolecule having a high binding affinity to norepinephrine was isolated from the soluble fraction of rat brain, the partially purified macromolecule was prepared by extraction of the brain with isotonic sucrose containing 3.3 mM CaCl2, ultracentrifugation, 35–50% ammonium sulfate precipitation and DEAE-cellulose chromatography. DEAE-cellulose chromatography revealed that the macromolecule in the soluble fraction was separated in at least 4 fractions, 2 of which had both norepinephrine and cyclic AMP binding activity. A Scatchard plot indicated that a partially purified macromolecule showed 2 apparent binding affinity constants of 4.13×10−7 M and 3.70×10−6 M to norepinephrine. The effect of chemical or physical treatments on specific binding of the macromolecule to norepinephrine was studied in comparison with cyclic AMP binding to the macromolecule. Trypsin treatment caused partial loss of norepinephrine binding activity, while cyclic AMP binding activity was completely lost. Heat treatment resulted in a complete disappearance of cyclic AMP binding activity, while norepinephrine binding activity was only slightly decreased. Dibenamine treatment caused a marked decrease both of norepinephrine binding activity and that of cyclic AMP. Dichloroisoproterenol had no effect on the binding activities. Cyclic AMP inhibited clearly norepinephrine binding to the macromolecule, while norepinephrine inhibited cyclic AMP binding only slightly. Norepinephrine binding to the macromolecule was essentially dependent on its catechol structure. The entity of the macromolecule is discussed on the basis of the findings.