Pig heart nucleosidediphosphate kinase Phosphorylation and interaction with Cibacron blue 3GA

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Abstract
The nucleoside diphosphate [NDP] kinase phosphorylation reaction led to the incorporation of 0.95 .+-. 0.1 phosphate groups per enzyme subunit. The equilibrium constant of the phosphorylation reaction was 0.26. The inhibition of the NDP kinase activity by Cibacron blue 3GA was competitive with respect to ATP, the donor nucleotide (apparent Ki = 0.28 .mu.M) and uncompetitive with respect to 8-bromoinosine 5''-diphosphate the acceptor nucleotide nucleotide (apparent Ki = 0.3 .mu.M). Difference spectroscopy showed that each enzyme subunit bound 1 Cibacron blue 3GA molecule, whereas the phosphorylated enzyme had no affinity for the dye. ATP was an effective competitor, being able to displace the dye from its bound state. The complex behavior noted was taken as evidence for cooperative interaction between the enzyme subunits. The data obtained using polarographic techniques agreed with these results.