Nucleoside pyrophosphatase activity associated with pig kidney alkaline phosphatase

1. A study was made of the hydrolysis, at pH9.0, of ATP and ADP catalysed by pig kidney alkaline phosphatase. Both of these nucleoside pyrophosphates are substrates for the enzyme; K(m) values are 4x10(-5)m for ATP and 6.3x10(-5)m for ADP. V(max.) for ADP is approximately double that of ATP. 2. Above 0.1mm approximately, both ATP and ADP are inhibitory, but the inhibition is reversible by the addition of Mg(2+) ions to form MgATP(2-) or MgADP(-) complexes. The complexes, besides being non-inhibitory, are also substrates for the enzyme with K(m) values identical with those of the respective free nucleotides. 3. Mg(2+) ions are inhibitory when present in excess of ATP or ADP. The degree of inhibition is greater with ATP as substrate, but with both ATP and ADP a mixed competitive-non-competitive type of inhibition is observed. 4. It is suggested that under normal conditions the enzyme is inhibited by cellular concentrations of ATP plus ADP but that an increase in the concentration of Mg(2+) ions stimulates activity by relieving nucleoside pyrophosphate inhibition. The properties may be of importance in the regulation of the transport of bivalent cations.     

Inhibition of pyrophosphatase activity of mouse duodenal alkaline phosphatase by magnesium ions

Duodenal alkaline phosphatase of juvenile (11-day-old) mice, like other non-specific alkaline phosphatases, has the ability to hydrolyse PP(i). When a constant Mg(2+)/PP(i) concentration ratio is maintained, plots of velocity as a function of PP(i) concentration are consistent with Michaelis-Menten kinetics. Mg(2+) activates pyrophosphate hydrolysis and maximal activity is obtained at a constant Mg(2+)/PP(i) concentration ratio of 0.66. At higher ratios there is strong inhibition. At constant concentrations of Mg(2+) and increasing concentrations of PP(i), the velocity-substrate (PP(i)) concentration plots show sigmoidal dependence. By assuming that the true substrate is MgP(2)O(7) (2-) complex, and using complexity constants, the concentrations of free Mg(2+), Mg(2)P(2)O(7) and MgP(2)O(7) (2-) were calculated in assay mixtures ranging in PP(i) concentration from 0.1 to 2.5mm and in total Mg(2+) concentration from 0.6 to 2.6mm. From these data, the concentrations of added Mg(2+) and PP(i) in the assay mixtures were selected so that the velocity could be measured (1) at three fixed concentrations of free Mg(2+) ions with varied concentrations of MgP(2)O(7) (2-) and (2) at four fixed concentrations of Mg(2)P(2)O(7) with varied concentrations of MgP(2)O(7) (2-). Lineweaver-Burk and Hill plots from these data showed that the inhibition is caused by free Mg(2+) ions, of a mixed type and consistent with Michaelis-Menten kinetics. The sigmoidal dependence observed between velocity and PP(i) concentration at constant concentration of total Mg(2+) is therefore not due to allosteric inhibition. It is due to a combined effect of (1) inhibition by free Mg(2+) ions, (2) depletion of the true substrate, MgP(2)O(7) (2-), owing to the formation of Mg(2)P(2)O(7) and (3) the manner in which the concentrations of these three molecular or ionic species change when PP(i) concentration is increased maintaining the total Mg(2+) concentration constant.     

Kinetics of alkaline phosphatase from pig kidney. Mechanism of activation by magnesium ions

The mechanism of activation of alkaline phosphatase (EC 3.1.3.1) from pig kidney by Mg²⁺ ions was investigated with the aid of kinetic measurements. Mg²⁺ ions are essential for enzyme activity. The following model (Scheme 1 of the text) for the reaction of enzyme, substrate and Mg²⁺ ions was derived: [Formula: see text] The binding of the substrate to the enzyme is independent of the binding of the activator, and vice versa. Mg²⁺ must therefore play a part in the substrate decomposition. It is not possible to determine whether the Mg²⁺ ions are involved directly in the catalytic process, or whether they act as regulatory effectors. Because of the strong affinity existing between the alkaline phosphatase and Mg²⁺, it is necessary to adjust the metal-ion concentration with the aid of a metal buffer. In the Appendix the necessary equations are derived for calculating the concentration of free metal ions in a system with several different metal ions. A FORTRAN IV program for solving these equations and for graphic presentation of the results has been deposited as Supplementary Publication SUP 50030 at the British Library (Lending Division) (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS 23 7 BQ, U.K., from whom copies may be obtained on the terms indicated in Biochem. J. (1973), 131, 5.     

Refolding and reactivation of calf intestinal alkaline phosphatase with excess magnesium ions

It is well known that Mg(2+) is an essential component in many biological processes. This research investigated the courses of both the reactivation and the refolding in the absence and presence of Mg(2+) ions. Calf intestinal alkaline phosphatase (CIP) was extensively denatured in 3 M guanidine hydrochloride (GdnHCl) solution for 2 h. Under suitable renaturation conditions, about 60-70% of the activity was recovered in the absence and presence of different magnesium ion concentrations. The refolding processes followed two-phase courses, whereas the reactivation processes were monophasic after dilution in proper solutions with or without Mg(2+). The magnesium ions affected both the reactivation and the refolding courses of unfolded CIP. A comparison of rate constants for the refolding of unfolded CIP with those for recovery of enzyme activity at different Mg(2+) concentrations showed that they were not synchronized. The activity recovery was speeded up due to the presence of Mg(2+) ions; while the refolding course of unfolded CIP was somewhat inhibited by the excess Mg(2+).     

Nicotinamide-adenine dinucleotide inhibition of pig kidney alkaline phosphatase.

1. The interaction of NAD+, NADH and various nucleotide analogues with pig kidney alkaline phosphatase (orthophosphoric-monoester phosphohydrolase (alkaline optimum) EC 3.1.3.1) has been investigated by kinetic means. Some inhibitors act uncompetitively whereas others markedly increase the slopes of double reciprocal plots suggesting they have some affinity for the free enzyme. 2. The compounds seem to bind to alkaline phosphatase through interactions of their bases with a relatively non-specific region of the enzyme, although it is likely that for those nucleotides having some affinity for the free enzyme there is some attraction between the pyrophosphate backbone and the active site. 3. From studies of the effect of NAD+ and NADH on ATPase activity it was concluded that the substrate inhibition that is characteristic of the ATPase activity of alkaline phosphatase originates from binding of ATP to the site assumed to exist for NAD+ and NADH. The potentiation of NAD+-inhibition of ATPase activity by Mg-2+ is probably a result of the depletion of [ATP-4-] the true substrate. The depletion allows NAD+ to complete more effectively for the active site. 4. Binding of NADH is favoured by protonation of an enzymic group with a pK of approx. 9.0 belonging possibly to a tyrosine residue or a zinc hydrate. 5. A large entropy decrease was found to accompany the binding of NAD+ and NADH to alkaline phosphatase. This may be further evidence of an "induced-fit" mechanism previously suspected because of the synergistic inhibitory effects of adenosine and nicotinamide.     

Effect of membrane moiety and magnesium ions on the inhibition of matrix-induced alkaline phosphatase by zinc ions

1. The inhibition of matrix-induced alkaline phosphatase by zinc ions is due to the displacement of magnesium ions from its binding site. 2. Binding of magnesium ions to alkaline phosphatase induces conformational changes which activate the enzyme. 3. Binding of zinc ions to alkaline phosphatase induces conformational changes which impair the catalytic action of the enzyme. 4. The inhibition of the enzyme by zinc ions is affected by membrane environment and magnesium ions.     

Alkaline inorganic pyrophosphatase activity of mammalian-cell alkaline phosphatase

Alkaline phosphatase prepared from mammalian cell cultures was found to have alkaline inorganic pyrophosphatase activity. Both of these activities appear to be associated with a single protein, as demonstrated by: (1) concomitant purification of alkaline phosphatase and alkaline inorganic pyrophosphatase; (2) proportional precipitation of alkaline phosphatase and inorganic pyrophosphatase activities by titrating constant amounts of an enzyme preparation with increasing concentration of antibody; (3) immune electrophoresis, which showed that precipitin bands that have alkaline phosphatase activity also have pyrophosphatase activity; (4) inhibition of pyrophosphatase activity by cysteine, an inhibitor of alkaline phosphatase activity; (5) similar subcellular localization of the two enzyme activities as demonstrated by histochemical methods; (6) hormonal and substrate induction of alkaline phosphatase activity in mammalian cell cultures, which produced a nearly parallel rise in inorganic pyrophosphatase activity.     

Catalytic properties of alkaline phosphatase from pig kidney

The enzymic properties of alkaline phosphatase (EC 3.1.3.1) from pig kidney brush-border membranes were studied. 1. It hydrolyses ortho- and pyro-phosphate esters, the rate limiting step (V(max.)) being independent of the substrate. It transphosphorylates to Tris at concentrations above 0.1m-Tris. 2. The pH optimum for hydrolysis was between 9.8 and 10. The pK of the enzyme-substrate complex is 8.7 for p-nitrophenyl phosphate and beta-glycerophosphate. Excess of substrate inhibits the enzymic activity with decreasing pH. The pK of the substrate-inhibited enzyme-substrate complex, 8.7, is very similar to that for the enzyme-substrate complex. The pK values of the free enzyme appear to be 8.7 and 7.9. 3. Inactivation studies suggest that there is an essential tyrosine residue at the active centre of the enzyme. 4. The energy of activation (E) and the heat of activation (DeltaH) at pH9.5 showed a transition at 24.8 degrees C that was unaffected by Mg(2+). 5. Kinetic and atomic-absorption analysis indicated the essential role of two Zn(2+) ions/tetrameric enzyme for an ordered association of the monomers. Zn(2+) in excess and other bivalent ions compete for a second site with Mg(2+). Mg(2+) enhances only the rate-limiting step of substrate hydrolysis. 6. Amino acid inhibition studies classified the pig kidney enzyme as an intermediate type of previously described alkaline phosphatases. It has more similarity with the enzyme from liver and bone than with that from placenta.     

Subcellular compartmentation of pyrophosphate and alkaline pyrophosphatase in leaves

The subcellular localisation of pyrophosphate and alkaline pyrophosphatase in leaves has been studied using non-aqueous density gradient centrifugation of spinach leaves, and membrane filtration of wheat mesophyll protoplasts. The pyrophosphate was measured in extracts prepared in trichloroacetic acid, and could be quantitatively recovered from the leaf material. It was located predominantly in the cytosol, with a concentration of 0.2–0.3 mM. In contrast, the alkaline pyrophosphatase was largely, if not, exclusively, located in the chloroplast. By comparing the pyrophosphate levels in the cytosol with previously published data on the cytosolic levels of phosphate and metabolic intermediates, it is shown that the reactions catalysed by pyrophosphate: fructose-6-phosphate phosphotransferase and UDP-glucose pyrophosphorylase are close to the thermodynamic equilibrium and, thus, freely reversible in vivo. Comparison of the pyrophosphate levels with the reported electrical and pH gradient across the tonoplast membrane shows the free energy released during pyrophosphate hydrolysis is similar to that required to move a proton across the tonoplast membrane. It is suggested that pyrophosphate could operate as a secondary energy donor in the cytosol of plant cells.     

The influence of metal ions on the orthophosphatase and inorganic pyrophosphatase activities of human alkaline phosphatase

1. The differential effects of adding Zn(2+) and Mg(2+) on the orthophosphatase and inorganic pyrophosphatase activities of human intestinal alkaline phosphatase were studied. 2. In the presence of excess of Zn(2+), inorganic pyrophosphatase activity is inhibited. At higher concentrations of pyrophosphate, hydrolysis of this substrate takes place, but is inhibited competitively by the Zn(2+)-pyrophosphate complex. This complex also acts as a competitive inhibitor of orthophosphate hydrolysis. 3. Excess of Mg(2+) also inhibits pyrophosphatase action by removal of substrate; at low concentrations, this ion activates pyrophosphatase, as is the case with orthophosphatase. 4. It is concluded that, when interactions between metal ions and pyrophosphate are taken into account, the effects of these ions are consistent with the view that alkaline phosphatases possess both orthophosphatase and inorganic pyrophosphatase activities.     

The reversible inactivation of pig kidney alkaline phosphatase at low pH

1. Pig kidney alkaline phosphatase is inactivated by treatment with acid at 0°. 2. Inactivated enzyme can be partially reactivated by incubation at 30° in neutral or alkaline buffer. The amount of reactivation that occurs depends on the degree of acid treatment; enzyme that has been inactivated below pH3·3 shows very little reactivation. 3. Studies of the kinetics of reactivation indicate that the process is greatly accelerated by increasing temperature and proceeds by a unimolecular mechanism. The reactivated enzyme has electrophoretic and gel-filtration properties identical with those of non-treated enzyme. 4. The results can be best explained by assuming that a lowering of the pH causes a reversible conformational change of the alkaline phosphatase molecule to a form that is no longer enzymically active but is very susceptible to permanent denaturation by prolonged acid treatment. A reactivation mechanism involving sub-unit recombination seems unlikely.