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

ABSTRACT: 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.

SUBMITTER: Rodan SB

PROVIDER: S-EPMC1161440 | biostudies-other | 1980 Mar

REPOSITORIES: biostudies-other

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Project description:The effects of a number of proteinase inhibitors on rat ovarian and rat hepatic adenylate cyclase preparations were examined. N alpha-tosylarginine methyl ester, 7-amino-1-chloro-3-L-tosylamidoheptan-2-one, 1-chloro-4-phenyl-3-L-tosylamidobutan-2-one, 1-chloro-4-methyl-3-L-tosylamidopentan-2-one and other low-molecular-weight proteinase inhibitors blocked hormonally stimulated adenylate cyclase from either source with hepatic preparations requiring higher concentrations. Addition of nucleotides (ATP, GTP, GDP, CTP or ITP) to inhibited ovarian preparations did not reverse inhibition, nor did dithiothreitol reverse phenylmethanesulphonyl fluoride-inhibited ovarian adenylate cyclase. The kinetics of the inhibition of rat ovarian adenylate cyclase were examined by following the production of cyclic AMP after the addition of inhibitors to membrane preparations preincubated under assay conditions with human choriogonadotropin, guanosine 5'-[beta gamma-imido]triphosphate of NaF. 7-Amino-1-chloro-3-L-tosylamidoheptan-2-one, 1-chloro-4-phenyl-3-L-tosylamidobutan-2-one and 1-chloro-4-methyl-3-L-tosylamidopentan-2-one had two effects on human-choriogonadotropin-stimulated adenylate cyclase. At low concentrations (less than or equal to 0.2 mM) there was an irreversible inhibition of hormonally-stimulated cyclase with maximum first-order inhibitory rate constants of 0.05--0.08 min-1. At higher concentrations the irreversible effect persisted, but, in addition, there was a marked decrease in the cyclase initial velocity to 25--50% of that of control values. N alpha-tosylarginine methyl ester had similar effects; at low concentrations (less than or equal to 2 mM) it inhibited irreversibly, and at higher concentrations it decreased the initial velocity (50% at 10 mM). At high concentrations (greater than 3 mM) N alpha-tosylarginine methyl ester also inhibited NaF- and guanosine 5'-[beta gamma-imidol]-triphosphate-stimulated cyclase but in a reversible manner. 7-Amino-1-chloro-3-L-tosylamidoheptan-2-one inhibited NaF-stimulated adenylate cyclase in two ways, as for human-choriogonadotropin-stimulated adenylate cyclase, but required 10--20-fold higher concentrations. The low-concentration irreversible effect can be explained by a continual inactive in equilibrium active conversion of adenylate cyclase during hormonal stimulation in which the inactive to active conversion is blocked by the inhibitors. The high-concentration effect is a direct one on the active catalytic moiety of the enzyme.

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

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