Comparison of bone and osteosarcoma adenylate cyclase. Effects of Mg2+, Ca2+, ATP4− and HATP3− in the assay mixture

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Abstract
The effects of Mg2+ and Ca2+ on bone and osteosarcoma adenylate cyclase were investigated. The concentrations of the cations and other ionic species in the assay mixture were calculated by solving the simultaneous equations describing the relevant ionic interactions (multiple equilibria). We re-examined the effects of HATP3− and ATP4− on enzyme activity and found that (i) the concentration of the minor ATP species is less than 1% of that of MgATP2−, and their ratio to MgATP2− is constant if Mg2+ and H+ concentrations are unchanged; (ii) Mg2+ addition decreased the ratio of the minor species to MgATP2− and increased the enzyme activity, but no meaningful kinetic model could attribute this effect of HATP3− or ATP4−. On the other hand, kinetic analysis of Mg2+ effects showed: (i) stimulation via two metal sites, separate from the catalytic (MgATP2−) site, with apparent Km values of approximately 1 and 8mm; (ii) that the low affinity increased towards the higher one when the enzyme activity rose as a result of increased substrate or guanine nucleotide concentrations, this effect being less pronounced in tumour; (iii) conversely, that two apparent affinities for MgATP2− merged into one at high Mg2+ concentration; (iv) kinetically, that this relationship is of the mixed con-competitive type, which is consistent with a role for Mg2+ as a requisite activator, and binding occurring in non-ordered sequence. Analysis of the Ca2+ effects showed: (i) competition with Mg2+ at the metal site (Ki 20μm for bone and 40μm for tumour); (ii) that relative to the substrate the inhibition was uncompetitive, i.e. velocity decreased and affinity increased proportionally, which is consistent with Ca2+ binding after substrate binding. These findings support the existence of interacting enzyme complexes, losing co-operativity at increased enzyme activity. They also indicate a potential physiological role for Ca2+ in enzyme regulation and point to quantitative differences between bone and tumour with regard to these properties.

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