Divalent cations and chelators as regulators of brain fructose-1,6-bisphosphatase

Abstract

Purified fish and rat brain FruP₂ase(s) are stimulated by a number of chelators, viz., histidine, EDTA, citrate, imidazole, and a number of histidine analogues. These also impart 5'-AMP sensitivity to the otherwise insensitive enzyme. Beyond 3 mM concentration, histidine inhibits the enzyme activity, which can be prevented by Mn²⁺. Atomic absorption spectrophotometry showed the presence of 5-6 mol of Mn²⁺ and Zn²⁺ bound to both fish and rat brain FruP₂ase, which can be removed by exhaustive EDTA-dialysis. The EDTA-dialyzed brain FruP₂ase records an absolute Mn²⁺ requirement and 5'-AMP sensitivity without any chelator treatment. The 5'-AMP sensitivity of such enzyme is abolished by prior incubation with Zn²⁺. The Zn²⁺-treated brain FruP₂ase fails to bind to a Blue-Sepharose column, in contrast to that seen using the untreated enzyme. These results suggest that rat and fish brain FruP₂ase(s) are actually Mn²⁺- and Zn²⁺-containing proteins with Zn²⁺ bound at or near the nucleotide-binding site.