The trigonal-bipyramidal NO4 ligand set in biologically relevant vanadium compounds and their inorganic models

In the present focused review, vanadate-dependent haloperoxidases and vanadate-inhibited enzymes which catalyze the hydrolysis of phosphoester bonds are addressed. In these systems, vanadate [H_xVO₄]^(3−x)− is covalently coordinated to the imidazolyl moiety of an active site histidine, with a geometrical arrangement close to a trigonal bipyramid. The resulting ligand set, NO₄, and ligand arrangement provide peroxidase activity to the haloperoxidases and, to a certain extent, also to vanadate-inhibited phosphatases. The haloperoxidases are responsible for the oxidative halogenation of a variety of organic substrates. They are also active in other oxidation reactions relying on peroxide as the oxidant, such as the oxidative cyclizations of terpenes and, specifically, the oxygenation of (prochiral) sulfides to (chiral) sulfoxides. These functions can be modeled by vanadium complexes. Attracted interest is paid to {V(NO₄)} complexes that are functional and structural models of the peroxidases. In the vanadate-inhibited phosphatases – structural analogs of the transition state in phosphoester hydrolysis by the native enzymes – the position of the axial histidine can also be taken by cysteinate or serinate, a fact which has implications for the insulin-mimetic potential of vanadate.     

The biosynthesis of peptidoglycan lipid-linked intermediates

The biosynthesis of bacterial cell wall peptidoglycan is a complex process involving many different steps taking place in the cytoplasm (synthesis of the nucleotide precursors) and on the inner and outer sides of the cytoplasmic membrane (assembly and polymerization of the disaccharide-peptide monomer unit, respectively). This review summarizes the current knowledge on the membrane steps leading to the formation of the lipid II intermediate, i.e. the substrate of the polymerization reactions. It makes the point on past and recent data that have significantly contributed to the understanding of the biosynthesis of undecaprenyl phosphate, the carrier lipid required for the anchoring of the peptidoglycan hydrophilic units in the membrane, and to the characterization of the MraY and MurG enzymes which catalyze the successive transfers of the N-acetylmuramoyl-peptide and N-acetylglucosamine moieties onto the carrier lipid, respectively. Enzyme inhibitors and antibacterial compounds interfering with these essential metabolic steps and interesting targets are presented.     

Probing the role of the divalent metal ion in uteroferrin using metal ion replacement and a comparison to isostructural biomimetics

Purple acid phosphatases (PAPs) are a group of heterovalent binuclear metalloenzymes that catalyze the hydrolysis of phosphomonoesters at acidic to neutral pH. While the metal ions are essential for catalysis, their precise roles are not fully understood. Here, the Fe(III)Ni(II) derivative of pig PAP (uteroferrin) was generated and its properties were compared with those of the native Fe(III)Fe(II) enzyme. The k _cat of the Fe(III)Ni(II) derivative (approximately 60 s⁻¹) is approximately 20% of that of native uteroferrin, and the Ni(II) uptake is considerably faster than the reconstitution of full enzymatic activity, suggesting a slow conformational change is required to attain optimal reactivity. An analysis of the pH dependence of the catalytic properties of Fe(III)Ni(II) uteroferrin indicates that the μ-hydroxide is the likely nucleophile. Thus, the Ni(II) derivative employs a mechanism similar to that proposed for the Ga(III)Zn(II) derivative of uteroferrin, but different from that of the native enzyme, which uses a terminal Fe(III)-bound nucleophile to initiate catalysis. Binuclear Fe(III)Ni(II) biomimetics with coordination environments similar to the coordination environment of uteroferrin were generated to provide both experimental benchmarks (structural and spectroscopic) and further insight into the catalytic mechanism of hydrolysis. The data are consistent with a reaction mechanism employing an Fe(III)-bound terminal hydroxide as a nucleophile, similar to that proposed for native uteroferrin and various related isostructural biomimetics. Thus, only in the uteroferrin-catalyzed reaction are the precise details of the catalytic mechanism sensitive to the metal ion composition, illustrating the significance of the dynamic ligand environment in the protein active site for the optimization of the catalytic efficiency. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Multiple Forms of Phosphatase from Human Brain: Isolation and Partial Characterization of Affi-Gel Blue Nonbinding Phosphatase Activities

Phosphatases extracted from a human brain were resolved into two main groups, namely affi-gel blue-binding phosphatases and affi-gel blue-nonbinding phosphatases. Affi-gel blue binding phosphatases were further separated into four different phosphatase activities, designated P1-P4, and described previously (1). In the present study we describe the affi-gel blue-nonbinding phosphatases which were separated into seven different phosphatase activities, designated P5-P11 by poly-(L-lysine)-agarose and aminohexyl Sepharose 4B chromatographies. These seven phosphatase activities were active toward nonprotein phosphoester. P7-P11 and to some extent P5 could also dephosphorylate a phosphoprotein. They displayed different enzyme kinetics. On the basis of activity peak, the apparent molecular mass as estimated by Sephadex G-200 column chromatography for P5 was 49 kDa; P6, 32 kDa; P7, 150 kDa; P8, 250 kDa; P9, 165 kDa; P10, 90 kDa and P11, 165 kDa. Immunoblot analysis indicated that P8-P11 may belong to PP2B family, whereas P7 may associate with PP2A. The phosphatases P7-P11 were found to be effective in the dephosphorylation of Alzheimer's disease abnormally hyperphosphorylated tau. The resulting dephosphorylated tau regained its activity in promoting the microtubule assembly, suggesting that P7-P11 might regulate the phosphorylation of tau protein in the brain.     

NMDA-Independent Long-Term Depression of Synaptic Transmission in the Hippocampus: Mechanisms of Induction and Effects of Nootropic Drugs

In studies on transversal slices of the rat dorsal hippocampus, we found that low-frequency tetanic stimulation of the medial perforant pathway (2 sec^-1, 7.5 min) results in long-term depression (LTD) of field EPSP of granular cells in the dentate gyrus. This synaptic plasticity phenomenon was weakened by calmodulin, nitric oxide synthase, and protein kinase C inhibitors, trifluoperazine (1 M), N-nitro-L-arginine (5 M), and polymixin B (50 M), respectively, but was enhanced by a nonselective inhibitor of cAMP phosphodiesterases, 1-isobuthyl-3-methylxanthine (100 M), and a calcineurin inhibitor, cyclosporin A (50 M). The nootropic activity-possessing drugs piracetam, carbacetam, and etimizole suppressed, in a dose-dependent manner, the induction and expression of the studied form of LTD of synaptic transmission, but glycine did not. We assume that Ca²⁺- and protein kinase G-mediated increase in the activity of calmodulin is the main link in the induction of this LTD form. Calmodulin, via NO synthase and adenylate cyclase, increases the activities of protein kinase C, a substrate of the latter, and inhibitor 1. Under the influence of piracetam, carbacetam, and etimizole, the calmodulin concentration in the cytoplasm of dendritic spines attains a level sufficient for activation of Ca²⁺/calmodulin-dependent protein kinase, which provides for the phosphorylation of AMPA receptors and interferes with the development of LTD of synaptic transmission.     

Tyrosine phosphorylation and morphological transfordmation induced by four vanadium compounds on MC3T3E1 cells

The present study was performed to determine the phosphotyrosine-protein levels induced by insulin and by four vanadium derivatives in MC3T3E1 osteoblast-like cells. We have also attempted to associate these patterns vath the vanadium-induced growth and morphological changes of such cells. Vanadate (Vi), vanadyl (VO), bis(maltolato)oxovanadium (IV) (BMOV) and bis(maltolato)dioxovanadium (V) (BMV) stimulate cell growth in a narrow range of concentration, but are also inhibitors for the cells at high concentrations. Vanadium-treated cells displayed clear changes in their morphology after overnight incubation. However, BMV was the least cytotoxic and the weakest inducer of morphological changes. All the compounds promote the phosphorylation of tyrosine residues in several proteins. This effect was more pronounced at low than at high doses. At low doses (10 M), BMV showed a phosphorylation pattern similar to that of insulin, while Vi, VO and BMOV induced strong phosphorylation of cell proteins. The present findings suggest that the vanadium-induced growth regulation and morphological changes in MC3T3EI osteoblast-like cells are associated with the ability of these agents to increase the phosphotyrosine protein levels and to inhibite phosphotyrosine phosphatases. These properties are dependent on the oxidation state as well as on the organic ligand which coordinates the vanadium atom.     

Dual fatty acylation of p59(Fyn) is required for association with the T cell receptor zeta chain through phosphotyrosine-Src homology domain-2 interactions

The first 10 residues within the Src homology domain (SH)-4 domain of the Src family kinase Fyn are required for binding to the immune receptor tyrosine-based activation motif (ITAM) of T cell receptor (TCR) subunits. Recently, mutation of glycine 2, cysteine 3, and lysines 7 and 9 was shown to block binding of Fyn to TCR zeta chain ITAMs, prompting the designation of these residues as an ITAM recognition motif (Gauen, L.K.T., M.E. Linder, and A.S. Shaw. 1996. J. Cell Biol. 133:1007-1015). Here we show that these residues do not mediate direct interactions with TCR ITAMs, but rather are required for efficient myristoylation and palmitoylation of Fyn. Specifically, coexpression of a K7,9A-Fyn mutant with N-myristoyltransferase restored myristoylation, membrane binding, and association with the cytoplasmic tail of TCR zeta fused to CD8. Conversely, treatment of cells with 2-hydroxymyristate, a myristoylation inhibitor, blocked association of wild-type Fyn with zeta. The Fyn NH2 terminus was necessary but not sufficient for interaction with zeta and both Fyn kinase and SH2 domains were required, directing phosphorylation of zeta ITAM tyrosines and binding to zeta ITAM phosphotyrosines. Fyn/zeta interaction was sensitive to octylglucoside and filipin, agents that disrupt membrane rafts. Moreover, a plasma membrane bound, farnesylated Fyn construct, G2A,C3S-FynKRas, was not enriched in the detergent insoluble fraction and did not associate with zeta. We conclude that the Fyn SH4 domain provides the signals for fatty acylation and specific plasma membrane localization, stabilizing the interactions between the Fyn SH2 domain and phosphotyrosines in TCR zeta chain ITAMs.     

Immunological relatedness of cytoplasmic and membrane-bound acid phosphatases from Dioscorea cayenensis rotundata

Antibodies were raised against one cytoplasmic and two membrane-bound acid phosphatases purified from yam tubers (Dioscorea cayenensis rotundata). Experiments of immunoinactivation and immunoelectrophoresis revealed cross-immunological reactions between the cytoplasmic enzyme (acid phosphatase A) and one of two membrane-bound counterparts (acid phosphatase B) suggesting that these molecules share common antigenic determinants. The antibodies raised against the other membrane-bound enzyme (acid phosphatase C) only inhibited and precipitated this enzyme.     

Reversible inhibition of the protein phosphatase 1 by hydrogen peroxide. Potential regulation of eIF2 alpha phosphorylation in differentiated PC12 cells

Oxidative inactivation of protein tyrosine phosphatases and calcineurin is a well established mechanism; however, little information with regard to the effect of oxidants on PP1 and PP2A activity is available. Herein, we show that PP1 activity is inhibited by H(2)O(2) treatment in differentiated PC12 cells both in vitro and in vivo experiments. Thiol-antioxidant N-acetyl-cysteine (NAC) and reduced glutathione (GSH), when added in vitro to lysates from H(2)O(2)-treated cells, reversed PP1 inhibition. H(2)O(2) treatment increased eIF2 alpha phosphorylated levels (eIF2 alpha P) in a time- and dose-dependent fashion and promoted protein synthesis inhibition. Interestingly, NAC pretreatment protected cells from H(2)O(2)-induced PP1 inactivation and, consequently, it abolished increased H(2)O(2)-induced eIF2 alpha phosphorylation and protein synthesis inhibition. In addition, PP1 inhibitor tautomycin prevented both NAC-induced PP1 reactivation and eIF2 alpha P dephosphorylation in H(2)O(2)-treated cells. Taken together, our findings support a role for PP1 in eIF2 alpha phosphorylation and oxidative stress-triggered translation down regulation.     

Does Spirodela punctata break P-C bonds?

Spirodela punctata was cultivated on phosphate-deficient medium (–P_i) with racemic 1-amino-2-phenylethylphosphonic acid (PheP) as a source of P_i. The growth of duckweed was inversely correlated with PheP concentration. The growth of plants on medium –P_i with 0.1 M PheP was accelerated whereas with 0.001 mM PheP was slower than in –P_i control. PheP at low concentrations decreased loss of chlorophyll in comparison with –P_i plants. Content of anthocyanins decreased but activity of the extractable constitutive phosphatases of pH 6.0 and pH 7.5 increased along with increasing concentration of PheP in the medium. We suggest that S. punctata does not break P-C bonds but probably PheP interrupts processes involved in the regulation of P_i-starvation response.