Dataset Information

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

ABSTRACT: 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.

SUBMITTER: Thorne MR

PROVIDER: S-EPMC1217305 | biostudies-other | 1996 May

REPOSITORIES: biostudies-other

ACCESS DATA

Similar Datasets

Project description:Synthesis of myo-inositol is crucial in multicellular eukaryotes for production of phosphatidylinositol and inositol phosphate signaling molecules. The myo-inositol monophosphatase (IMP) enzyme is required for the synthesis of myo-inositol, breakdown of inositol (1,4,5)-trisphosphate, a second messenger involved in Ca(2+) signaling, and synthesis of L-galactose, a precursor of ascorbic acid. Two myo-inositol monophosphatase -like (IMPL) genes in Arabidopsis encode chloroplast proteins with homology to the prokaryotic IMPs and one of these, IMPL2, can complement a bacterial histidinol 1-phosphate phosphatase mutant defective in histidine synthesis, indicating an important role for IMPL2 in amino acid synthesis. To delineate how this small gene family functions in inositol synthesis and metabolism, we sought to compare recombinant enzyme activities, expression patterns, and impact of genetic loss-of-function mutations for each. Our data show that purified IMPL2 protein is an active histidinol-phosphate phosphatase enzyme in contrast to the IMPL1 enzyme, which has the ability to hydrolyze D-galactose 1-phosphate, and D-myo-inositol 1-phosphate, a breakdown product of D-inositol (1,4,5) trisphosphate. Expression studies indicated that all three genes are expressed in multiple tissues, however, IMPL1 expression is restricted to above-ground tissues only. Identification and characterization of impl1 and impl2 mutants revealed no viable mutants for IMPL1, while two different impl2 mutants were identified and shown to be severely compromised in growth, which can be rescued by histidine. Analyses of metabolite levels in impl2 and complemented mutants reveals impl2 mutant growth is impacted by alterations in the histidine biosynthesis pathway, but does not impact myo-inositol synthesis. Together, these data indicate that IMPL2 functions in the histidine biosynthetic pathway, while IMP and IMPL1 catalyze the hydrolysis of inositol- and galactose-phosphates in the plant cell.

Project description:We describe a hybrid metal-dielectric waveguide structures (MDWs) with numerous potential applications in the biosciences. These structures consist of a thin metal film coated with a dielectric layer. Depending on the thickness of the dielectric layer, the modes can be localized near the metal, within the dielectric, or at the top surface of the dielectric. The optical modes in a metal-dielectric waveguide can have either S (TE) or P (TM) polarization. The dielectric spacer avoids the quenching, which usually occurs for fluorophores within about 5 nm from the metal. Additionally, the resonances display a sharp angular dependence and can exhibit several hundred-fold increases in intensity (E2) at the silica-air interface relative to the incident intensity. Fluorophores placed on top of the silica layer couple efficiently with the metal, resulting in a sharp angular distribution of emission through the metal and down from the bottom of the structure. This coupling occurs over large distances to several hundred nm away from the metal and was found to be consistent with simulations of the reflectivity of the metal-dielectric waveguides. Remarkably, for some silica thicknesses, the emission is almost completely coupled through the structure with little free-space emission away from the metal-dielectric waveguide. The efficiency of fluorophore coupling is related to the quality of the resonant modes sustained by the metal-dielectric waveguide, resulting in coupling of most of the emission through the metal into the underlying glass substrates. Metal-dielectric waveguides also provide a method to resolve the emission from surface-bound fluorophores from the bulk-phase fluorophores. Metal-dielectric waveguides are simple to fabricate for large surface areas, the resonance wavelength can be adjusted by the dielectric thickness, and the silica surface is suitable for coupling to biomolecules. Metal-dielectric waveguides can have numerous applications in diagnostics and high-throughput proteomics or DNA analysis.

Dataset Information

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets