Metal and metal-ATP interactions with brain and cardiac adenylate cyclases.

Garbers, D L; Johnson, RA

doi:10.1016/s0021-9258(19)40779-5

Cited by 142 publications

(9 citation statements)

References 27 publications

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“…However, we found no support for the hypothesis that other ATP species in equilibrium with MgATP2inhibit the enzyme activity (deHaen, 1974;Rendell et al, 1975). These findings confirm the conclusions of Garbers & Johnson (1975) and Londos & Preston (1977).…”

Section: Discussionsupporting

confidence: 91%

“…We have shown in the preceding paper (Rodan et al, 1980) that rat osteosarcoma membranes possess significantly lower adenylate cyclase activity, probably as the result of differences in enzyme regulation. One of the major modulators of enzyme activity is Mg2+, which, in addition to its participation in substrate formation (MgATP2-), controls enzyme activity by acting as an obligatory activator at a separate site (Drummond & Duncan, 1970;Garbers & Johnson, 1975;Londos & Preston, 1977; Abbreviation used: p[NH]ppG, guanosine 5'-[flyimidoltriphosphate.…”

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confidence: 99%

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

Rodan

Golub

Egan

et al. 1980

Biochemical Journal

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The effects of Mg(2+) and Ca(2+) 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 HATP(3-) and ATP(4-) on enzyme activity and found that (i) the concentration of the minor ATP species is less than 1% of that of MgATP(2-), and their ratio to MgATP(2-) is constant if Mg(2+) and H(+) concentrations are unchanged; (ii) Mg(2+) addition decreased the ratio of the minor species to MgATP(2-) and increased the enzyme activity, but no meaningful kinetic model could attribute this effect of HATP(3-) or ATP(4-). On the other hand, kinetic analysis of Mg(2+) effects showed: (i) stimulation via two metal sites, separate from the catalytic (MgATP(2-)) site, with apparent K(m) 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 MgATP(2-) merged into one at high Mg(2+) concentration; (iv) kinetically, that this relationship is of the mixed con-competitive type, which is consistent with a role for Mg(2+) as a requisite activator, and binding occurring in non-ordered sequence. Analysis of the Ca(2+) effects showed: (i) competition with Mg(2+) at the metal site (K(i) 20mum for bone and 40mum for tumour); (ii) that relative to the substrate the inhibition was uncompetitive, i.e. velocity decreased and affinity increased proportionally, which is consistent with Ca(2+) 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 Ca(2+) in enzyme regulation and point to quantitative differences between bone and tumour with regard to these properties.

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Section: Discussionsupporting

confidence: 91%

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confidence: 99%

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

Rodan

Golub

Egan

et al. 1980

Biochemical Journal

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“…Effect of Mg 2+ and Mn 2+ on the Labeling of IIC2 by oATP and by FITC. For enzymatic activity, adenylyl cyclase requires a divalent cation, Mg 2+ being presumably the physiological ligand; however, Mn 2+ efficiently substitutes for Mg 2+ and increases the rate of catalysis (1,11). We have therefore examined whether oATP binding to IIC2 depends on the presence of a divalent cation.…”

Section: Resultsmentioning

confidence: 99%

“…The recently solved crystal structure of the complex of a C1 and a C2 domain indicates that the ATP binding site is formed by surfaces that are in the cleft between the two domains; in addition, a single Mg 2+ was visualized in the crystal of the C1-C2 heterodimer, and this was liganded to two aspartate residues of C1 (9). However, adenylyl cyclase requires Mg 2+ or Mn 2+ in excess of ATP for catalysis (11). Moreover, the active site of DNA polymerases contains two metal ions termed A and B (26-28); metal ion A presumably polarizes the 3′-OH group, and metal ion B interacts with the three phosphates of the incoming nucleotide and is likely to correspond to the Mg 2+ present in the C1-C2 heterodimer (4).…”

Section: Discussionmentioning

confidence: 99%

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The C2 Catalytic Domain of Adenylyl Cyclase Contains the Second Metal Ion (Mn²⁺) Binding Site

et al. 1998

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Membrane-bound mammalian adenylyl cyclase isoforms contain two internally homologous cytoplasmic domains (C1 and C2). When expressed separately, C1 and C2 are catalytically inactive, but conversion of ATP to cAMP is observed if C1 and C2 are combined. By analogy with DNA polymerases, adenylyl cyclases are thought to require two divalent metal ions for nucleotide binding and phosphodiester formation; however, only one Mg2+ ion (liganded to C1) has been visualized in the recently solved crystal structure of a C1-C2 complex [Tesmer, J. J. G., Sunahara, R. K., Gilman, A. G., and Sprang, S. R. (1997) Science 278, 1907-1916]. Here, we have studied the binding of ATP to IIC2 (from type II adenylyl cyclase) using ATP analogues [2',3'-dialdehyde ATP (oATP), a quasi-irreversible inhibitor that is covalently incorporated via reduction of a Schiff base, the photoaffinity ligand 8-azido-ATP (8N3-ATP), and trinitrophenyl-ATP (TNP-ATP), a fluorescent analogue] and fluorescein isothiocyanate (FITC). [alpha-32P]oATP and 8N-[alpha-32P]ATP are specifically incorporated into IIC2. Labeling of IIC2 by [alpha-32P]oATP and by FITC is greatly enhanced by Mn2+ and to a much lesser extent by Mg2+. Similarly, TNP-ATP binds to IIC2 as determined by fluorescence enhancement, and this binding is promoted by Mn2+. Thus, a second metal ion binding site (preferring Mn2+) is contained within the C2 domain, and this finding highlights the analogy in the reaction catalyzed by DNA polymerases and adenylyl cyclases.

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Interaction of manganese with NB41A neuroblastoma adenylate cyclase

Reiss

Sapirstein

1983

J of Neuroscience Research

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In vitro assay of the adenylate cyclase of NB41A neuroblastoma cells in the presence of increasing concentrations of MnCl2 suggested that the enzyme is modulated by both high- and low-affinity sites for manganese. MnCl2 in a concentration of 1 microM significantly stimulated adenylate cyclase activity, but increasing the concentration of manganese to 3 microM or 10 microM had no further effect. Raising MnCl2 to 0.1 or 1 mM, however, further stimulated enzyme activity. In addition to differences in affinity for manganese, the two classes of binding sites may be distinguished by differences in their interaction with other agents that affect adenylate cyclase activity. Millimolar manganese and magnesium appeared to compete for a common site on the enzyme and the effect of manganese in this range and the effect of guanyl nucleotide were synergistic. In contrast, the stimulation of activity by micromolar manganese appeared to be additive to the effects of either increasing magnesium or the addition of guanyl nucleotide to the assay media. Comparison of the substrate dependency of the reaction measured in the presence and absence of manganese suggests that the stimulation of adenylate cyclase activity involves increases in both the apparent Vmax of the reaction and the affinity for ATP. The results raise the possibility that the interaction of Mn2+ may play a role in the modulation of adenylate cyclase in vivo.

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Metal and metal-ATP interactions with brain and cardiac adenylate cyclases.

Cited by 142 publications

References 27 publications

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

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

The C2 Catalytic Domain of Adenylyl Cyclase Contains the Second Metal Ion (Mn²⁺) Binding Site

Interaction of manganese with NB41A neuroblastoma adenylate cyclase

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Metal and metal-ATP interactions with brain and cardiac adenylate cyclases.

Cited by 142 publications

References 27 publications

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

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

The C2 Catalytic Domain of Adenylyl Cyclase Contains the Second Metal Ion (Mn2+) Binding Site

Interaction of manganese with NB41A neuroblastoma adenylate cyclase

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The C2 Catalytic Domain of Adenylyl Cyclase Contains the Second Metal Ion (Mn²⁺) Binding Site