Bovine inositol monophosphatase: proteolysis and structural studies

Greasley, Peter J.; Gore, Michael G.; Rees-Milton, Karen; Ragan, C I

doi:10.1016/0014-5793(93)80035-s

Cited by 5 publications

(5 citation statements)

References 23 publications

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“…Since the K,,, for the metal ion is dependent upon the substrate used, these studies are the first to monitor the fundamental metal-protein interactions. Limited proteolysis by trypsin has been demonstrated to cleave the peptide bond between Lys36 and Ser37, effectively, removing a peptide element that is thought to move during catalysis [24]. Previous studies have shown that this treatment eliminates the activity of the enLyme and fluorescence studies have indirectly supported the proposal that this is probably achieved by also eliminating the binding of Mg" to site 1.…”

Section: Discussionmentioning

confidence: 76%

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Detection of Metal Binding to Bovine Inositol Monophosphatase by Changes in the Near and Far Ultraviolet Regions of the CD Spectrum

Rees-Milton

Thorne

Greasley

et al. 1997

European Journal of Biochemistry

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Mg" ions, essential for the catalytic activity of mammalian inositol monophosphatase, increase the ellipticity in the near-ultraviolet region of the CD spectrum of the enzyme. These spectral changes are not affected by the additional presence of substrate and are reversed if EDTA is added to the solution of enzyme and metal ions. Titration of the spectral perturbation at 275 nm shows that this binding occurs with a dissociation constant ( K J around 275 pM, 292 pM and 302 pM for the wild-type, [Gln217]inositol monophosphatase and [Phe219]inositol monophosphatase enzymes respectively. The source of the spectroscopic change at 275 nm is not Trp219.The addition of Mgz+ also causes a decrease in ellipticity over most of the far-ultraviolet region of the spectrum (between 205-240 nm). The Kd values describing the binding of Mg" ions are 3.9 mM, 6.8 mM and 29.1 mM for the wild-type, [Gln217]inositol monophosphatase and [Phe219]inositol monophosphatase enzymes, respectively, each showing an approximate 12 % change in ellipticity. In the additional presence of 10 mM P,, there is a fourfold increase in the affinity of wild-type enzyme for Mg".It is concluded that CD spectral changes at wavelengths around 275 nm are indicative of metal ions interacting with a high-affinity metal-binding site (site 3 ) . The spectral changes around 225 nm are associated with interactions at a lower-affinity site normally occupied by the Mg2' ion which is reflected by the K,, value for this metal ion.Other metal ions such as Ca2+ and Tb" (but not Mn'+ or Znz+) also perturb the CD spectrum of the enzyme in both regions of the spectrum. The amplitudes of these signal changes are greater for Mg" or Tb" (25%) ions than for Ca2+ (8.5%), although two Caz+-binding sites with Kd values of 20 pM and 100 pM have been identified.The uncompetitive inhibitor Li' causes little change in the near-ultraviolet spectrum in the absence or presence of either substrate or P,. However, in contrast to other metal ions, Li' ions elicit a 10% increase in ellipticity at 220 nm with a Kd of 0.8 mM.Keywords: inositol monophosphatase ; circular dichroism; lithium ; magnesium.Inositol monophosphatase is a homodimeric enzyme of subunit molecular mass 30 kDa that catalyses the dephosphorylation of Ins( 1)P, Ins(3)P and Ins(4)P (inositol monophosphates) to liberate inositol and P, (inorganic phosphate), the former of which may be used to replenish stores of membrane-bound phosphatidylinositol 4,5-bisphosphate [l]. The pivotal role in the phosphoinositol lipid signalling pathway played by this enzyme is of greater significance in the brain due to the inability of dietary inositol to cross the blood-brain barrier effectively 121, although inositol may be synthesised de novo from glucose 6-phosphate via the action of inositol 1-synthase [3]. A high level of sequence similarity between the human, rat and bovine forms of the enzyme has been demonstrated using cDNA cloning and sequencing techniques [4, 51. Mg2' ions are absolutely required for activity [6], although concentratio...

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Section: Discussionmentioning

confidence: 76%

“…To 1 ml of inosito1 monophosphatase (1 mg/ml) was added 20 pl of 0.1 mg/ml trypsin in 10 mM Tris/HCl, pH 8.0, at 21 "C. Samples were withdrawn at various times and either assayed for activity [22] or run on a 12% SDYPAGE gel for verification that a single cleavage of the protein had occurred [24].…”

Section: Methodsmentioning

confidence: 99%

Detection of Metal Binding to Bovine Inositol Monophosphatase by Changes in the Near and Far Ultraviolet Regions of the CD Spectrum

Rees-Milton

Thorne

Greasley

et al. 1997

European Journal of Biochemistry

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“…A sample of the pyrene-labelled enzyme was subjected to partial proteolysis by trypsin, previously shown to cleave specifically between Lys-36 and Ser-37 [25]. Such treatment leads to the loss of a 36-amino-acid peptide from the N-terminal end of the protein, resulting in complete loss of enzyme activity [25]. Stopped-flow fluorescence studies using these enzyme species showed that neither the fast nor the slow change in fluorescence intensity occurred from the pyrene moiety on mixing with Mg# + ions.…”

Section: Effect Of Limited Proteolysis By Trypsin On Both Phases Of Tmentioning

confidence: 99%

“…The epitope of the antibody is centred around Cys-8 [23], and binding gives rise to inhibition of the enzyme [23]. Furthermore, limited proteolysis studies using endoproteinase Lys-C [24] and trypsin [25] have demonstrated that the bond between Lys-36 and Ser-37 in inositol monophosphatase is highly susceptible to proteolysis. Hydrolysis of this bond leads to a concurrent loss of enzyme activity, suggesting that this region of the protein is important in the functioning of the enzyme.…”

Section: Introductionmentioning

confidence: 99%

“…Hydrolysis of this bond leads to a concurrent loss of enzyme activity, suggesting that this region of the protein is important in the functioning of the enzyme. The susceptibility of this bond to cleavage is decreased by the presence of substrate or other ligands known to bind at the active site of the enzyme [24,25], suggesting that a conformational rearrangement occurs in this region after the ligands have bound.…”

Section: Introductionmentioning

confidence: 99%

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Bovine inositol monophosphatase: enzyme—metal-ion interactions studied by pre-equilibrium fluorescence spectroscopy

Thorne

Greasley

Gore

1996

Biochemical Journal

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Stopped-flow fluorescence spectroscopy has been used to determine the on-rate (kass) and the off-rate (kdiss) for the equilibrium between inositol monophosphatase and Mg2+ ions. The dissociation constant (Kd) for the equilibrium calculated from these constants suggests that the ions interact at site 1 on the enzyme with a Kd typically around 450 microM, close to values determined by equilibrium studies (270-300 microM). The affinity of this site on the wild-type enzyme for Mg2+ ions increases as the pH is increased. This is mediated almost entirely by change in the rate kdiss. A slow increase occurs in the fluorescence intensity of the pyrene-labelled enzyme after the initial, fast, increase in fluorescence caused by the binding of the Mg2+ ion. The rate of this change is independent of the concentration of the metal ion, implying that it may be a structural change in the enzyme-Mg2+ complex. Neither the fast nor the slow change in fluorescence intensity occurs when enzyme subjected to limited proteolysis by trypsin, which removes the N-terminal 36 residues, is mixed with Mg2+ ions. The data suggest that interaction with Mg2+ ions at a high-affinity site leads to a structural change in inositol monophosphatase. The data further confirm the importance of the presence of two metal ions in the structure/function of this enzyme, and show that the binding of the metal ions is not competitive with that of H+ ions and that the variation in Kd with pH is mediated almost totally by changes in kdiss.

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Bovine inositol monophosphatase

Greasley¹,

Gore²

1993

FEBS Letters

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Rapid equilibrium dialysis has been used to show that recombinant bovine brain inositol monophosphatase binds one equivalent of Pi per subunit of enzyme. Pi is only bound in the presence of Mg2+ ions. The dissociation constant for the equilibrium is approximately 50 μM. This value of K d is independent of the concentration of the Mg2+ ions and of the presence or absence of Li+ ions. Lithium ions which inhibit the enzyme uncompetitively are not able to support the binding of the Pi to the enzyme. The observation that Pi only binds in the presence of Mg2+ ions supports similar conclusions made in experiments which studied the protection of the enzyme from proteolytic degradation and chemical modification.

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Bovine inositol monophosphatase: proteolysis and structural studies

Cited by 5 publications

References 23 publications

Detection of Metal Binding to Bovine Inositol Monophosphatase by Changes in the Near and Far Ultraviolet Regions of the CD Spectrum

Detection of Metal Binding to Bovine Inositol Monophosphatase by Changes in the Near and Far Ultraviolet Regions of the CD Spectrum

Bovine inositol monophosphatase: enzyme—metal-ion interactions studied by pre-equilibrium fluorescence spectroscopy

Bovine inositol monophosphatase

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