1. An inositol monophosphatase was purified to homogeneity from bovine brain. 2. The enzyme is a dimer of subunit Mr 29,000. 3. The enzyme hydrolyses both enantiomers of myo-inositol 1-phosphate and both enantiomers of myo-inositol 4-phosphate, but has no activity towards inositol bisphosphates, inositol trisphosphates or inositol 1,3,4,5-tetrakisphosphate. 4. Several non-inositol-containing monophosphates are also substrates. 5. The enzyme requires Mg2+ for activity, and Zn2+ supports activity to a small extent. 6. Other bivalent cations (including Zn2+) are inhibitors, competitive with Mg2+. 7. Phosphate, but not inositol, is an inhibitor competitive with substrate. 8. Li+ inhibits hydrolysis of inositol 1-phosphate and inositol 4-phosphate uncompetitively with different apparent Ki values (1.0 mM and 0.26 mM respectively).
Lithium-sensitive inositol monophosphatase from bovine brain was purified from brain and from a recombinant strain of Escherichia coli BL21-DE3. The natural and recombinant enzymes displayed identical physical and kinetic properties. At low [Li'], Li' inhibited the hydrolysis of racemic myo-inositol 1 -phosphate, myo-inositol4-phosphate and adenosine 2'-phosphate in a linear uncompetitive manner with apparent K, values of 1.1, 0.11 and 1.52mM, respectively. At Li' concentrations higher than 4 mM, Li' acted as a non-linear noncompetitive inhibitor for myo-inositol 1-phosphate, K, greater than 1.5 mM.The enzyme was unable to catalyze the transesterification of ['4C]inositol in the presence of inositol 1-phosphate or adenosine 2'-phosphate and attempts to trap a phosphorylated enzyme intermediate directly, were unsuccessful. In the presence of Li+, the enzyme was able to release inositol from inositol 1-phosphate, in a burst, faster than the rate of steady-state substrate turnover suggesting that Li' binds after P-0 bond cleavage in the substrate has occurred. The possibility that a free phosphorylated enzyme intermediate might exist was discounted when the exchange of "0 from water into phosphate was shown to be completely dependent upon inositol. The K, for inositol for "0 exchange was 190 mM and in the presence of saturating phosphate, VEX was at least 60% of V,, for the hydrolysis reaction. Thus, the enzyme operates via a ternary-complex mechanism, and Li' exerts its action by binding to enzyme/product complexes. At low concentration, Li' inhibition with respect to the cofactor, Mg2+ was non-competitive. Mg2+ acted as a non-competitive activator for substrate hydrolysis at pH 8.0, but as the second substrate in an equilibrium-ordered mechanism at pH 6.5. Cooperativity effects were observed for Mg2+ with inositol I-phosphate and 2'AMP as the substrate but not with inositol4-phosphate. The combined results indicate that Mgz' and substrate binding is ordered with substrate adding first. Inositol, the first product off, was a poor non-competitive inhibitor for inositol 1-phosphate whereas the other product, phosphate, was a competitive inhibitor. Phosphate inhibition was markedly pH dependent (K, = 8 mM at pH 6.5 and 0.32 mM at pH 8.0). In the presence of Li' and phosphate, increasing [Li'] caused the K, for phosphate to decrease by a factor of (1 + [Li+]/K,,). The K, for the first product off (inositol) was, however, unaltered by Li'. The results indicate that Li' can bind to the species E.Ins.P, and E.P,, but not to enzyme/substrate complexes. Further examination of the burst-phase release of [14C]inositol and its rate relative to that of the steady-state reaction under a variety of conditions revealed that Li' acts as a retarder rather than as a dead-end inhibitor and that the burst was due to hysteresis.Evidence is provided to suggest that Mg2+ is required for the catalysis only and that Li' occupies the site vacated by Mg2+ in its action as an inhibitor. The mechanisms of the reactions, the modes of inhibition...
Nitrogen dioxide (NO2) is a free radical and a common oxidant in polluted air. Here we present data on the time course of inflammation after NO2 exposure, as reflected in bronchial biopsy and airway lavage specimens. Healthy, nonsmoking subjects were exposed to air or 2 ppm NO2 for 4 h in random order on separate occasions. Endobronchial biopsies, bronchial washing (BW), and bronchoalveolar lavage (BAL) were done at 1.5 h (n = 15) or 6 h (n = 15) after exposure. In BW, exposure to NO2 induced a 1.5-fold increase in interleukin-8 (IL-8) (p < 0.05) at 1.5 h and a 2.5-fold increase in neutrophils (p < 0.01) at 6 h. In BAL fluid (BALF), small increases were observed in CD45RO+ lymphocytes, B-cells, and natural killer (NK) cells only. Immunohistologic examination of bronchial biopsy specimens showed no signs of upregulation of adhesion molecules, and failed to reveal any significant changes in inflammatory cells at either time point after NO2 exposure. In summary, NO2 induced a neutrophilic inflammation in the airways that was detectable in BW at 6 h after NO2 exposure. The increase in neutrophils could be related to the enhanced IL-8 secretion observed at 1.5 h after exposure. The absence of adhesion-molecule upregulation or cellular inflammation in mucosal biopsy specimens indicates that the major site of inflammation following exposure to NO2 may be in the smaller airways and not in the alveoli.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.