Cloning and characterization of a cDNA encoding a novel extracellular peroxidase from Trametes versicolor.
The white rot basidiomycete Trametes versicolor secretes a large number of peroxidases which are believed to be involved in the degradation of polymeric lignin. These peroxidases have been classified previously as lignin peroxidases or manganese peroxidases (MnP). We have isolated a novel extracellular peroxidase-encoding cDNA sequence from T. versicolor CU1, the transcript levels of which are repressed by low concentrations of Mn2+ and induced by nitrogen and carbon but not induced in response to a range of stresses which have been reported to induce MnP expression. (+info)
A functional model for O-O bond formation by the O2-evolving complex in photosystem II.
The formation of molecular oxygen from water in photosynthesis is catalyzed by photosystem II at an active site containing four manganese ions that are arranged in di-mu-oxo dimanganese units (where mu is a bridging mode). The complex [H2O(terpy)Mn(O)2Mn(terpy)OH2](NO3)3 (terpy is 2,2':6', 2"-terpyridine), which was synthesized and structurally characterized, contains a di-mu-oxo manganese dimer and catalyzes the conversion of sodium hypochlorite to molecular oxygen. Oxygen-18 isotope labeling showed that water is the source of the oxygen atoms in the molecular oxygen evolved, and so this system is a functional model for photosynthetic water oxidation. (+info)
Manganese sulfate-dependent glycosylation of endogenous glycoproteins in human skeletal muscle is catalyzed by a nonglucose 6-P-dependent glycogen synthase and not glycogenin.
Glycogenin, a Mn2+-dependent, self-glucosylating protein, is considered to catalyze the initial glucosyl transfer steps in glycogen biogenesis. To study the physiologic significance of this enzyme, measurements of glycogenin mediated glucose transfer to endogenous trichloroacetic acid precipitable material (protein-bound glycogen, i.e., glycoproteins) in human skeletal muscle were attempted. Although glycogenin protein was detected in muscle extracts, activity was not, even after exercise that resulted in marked glycogen depletion. Instead, a MnSO4-dependent glucose transfer to glycoproteins, inhibited by glycogen and UDP-pyridoxal (which do not affect glycogenin), and unaffected by CDP (a potent inhibitor of glycogenin), was consistently detected. MnSO4-dependent activity increased in concert with glycogen synthase fractional activity after prolonged exercise, and the MnSO4-dependent enzyme stimulated glucosylation of glycoproteins with molecular masses lower than those glucosylated by glucose 6-P-dependent glycogen synthase. Addition of purified glucose 6-P-dependent glycogen synthase to the muscle extract did not affect MnSO4-dependent glucose transfer, whereas glycogen synthase antibody completely abolished MnSO4-dependent activity. It is concluded that: (1) MnSO4-dependent glucose transfer to glycoproteins is catalyzed by a nonglucose 6-P-dependent form of glycogen synthase; (2) MnSO4-dependent glycogen synthase has a greater affinity for low molecular mass glycoproteins and may thus play a more important role than glucose 6-P-dependent glycogen synthase in the initial stages of glycogen biogenesis; and (3) glycogenin is generally inactive in human muscle in vivo. (+info)
Suppression of a nuclear aep2 mutation in Saccharomyces cerevisiae by a base substitution in the 5'-untranslated region of the mitochondrial oli1 gene encoding subunit 9 of ATP synthase.
Mutations in the nuclear AEP2 gene of Saccharomyces generate greatly reduced levels of the mature form of mitochondrial oli1 mRNA, encoding subunit 9 of mitochondrial ATP synthase. A series of mutants was isolated in which the temperature-sensitive phenotype resulting from the aep2-ts1 mutation was suppressed. Three strains were classified as containing a mitochondrial suppressor: these lost the ability to suppress aep2-ts1 when their mitochondrial genome was replaced with wild-type mitochondrial DNA (mtDNA). Many other isolates were classified as containing dominant nuclear suppressors. The three mitochondrion-encoded suppressors were localized to the oli1 region of mtDNA using rho- genetic mapping techniques coupled with PCR analysis; DNA sequencing revealed, in each case, a T-to-C nucleotide transition in mtDNA 16 nucleotides upstream of the oli1 reading frame. It is inferred that the suppressing mutation in the 5' untranslated region of oli1 mRNA restores subunit 9 biosynthesis by accommodating the modified structure of Aep2p generated by the aep2-ts1 mutation (shown here to cause the substitution of proline for leucine at residue 413 of Aep2p). This mode of mitochondrial suppression is contrasted with that mediated by heteroplasmic rearranged rho- mtDNA genomes bypassing the participation of a nuclear gene product in expression of a particular mitochondrial gene. In the present study, direct RNA-protein interactions are likely to form the basis of suppression. (+info)
Extracellular heavy-metal ions stimulate Ca2+ mobilization in hepatocytes.
Populations of hepatocytes in primary culture were loaded with fura 2 and the effects of extracellular heavy-metal ions were examined under conditions that allowed changes in fura 2 fluorescence (R340/360, the ratio of fluorescence recorded at 340 and 360 nm) to be directly attributed to changes in cytosolic free [Ca2+] ([Ca2+]i). In Ca2+-free media, Ni2+ [EC50 (concentration causing 50% stimulation) approximately 24+/-9 microM] caused reversible increases in [Ca2+]i that resulted from mobilization of the same intracellular Ca2+ stores as were released by [Arg8]vasopressin. The effects of Ni2+ were not mimicked by increasing the extracellular [Mg2+], by addition of MnCl2, CoCl2 or CdCl2 or by decreasing the extracellular pH from 7.3 to 6.0; nor were they observed in cultures of smooth muscle, endothelial cells or pituitary cells. CuCl2 (80 microM), ZnCl2 (80 microM) and LaCl3 (5 mM) mimicked the ability of Ni2+ to evoke Ca2+ mobilization. The response to La3+ was sustained even in the absence of extracellular Ca2+, probably because La3+ also inhibited Ca2+ extrusion. Although Ni2+ entered hepatocytes, from the extent to which it quenched fura 2 fluorescence the free cytosolic [Ni2+] ([Ni2+]i) was estimated to be <5 nM at the peak of the maximal Ni2+-evoked Ca2+ signals and there was no correlation between [Ni2+]i and the amplitude of the evoked increases in [Ca2+]i. We conclude that extracellular Ni2+, Zn2+, Cu2+ and La3+, but not all heavy-metal ions, evoke an increase in [Ca2+]i in hepatocytes by stimulating release of the hormone-sensitive intracellular Ca2+ stores and that they may do so by interacting with a specific cell-surface ion receptor. This putative ion receptor may be important in allowing hepatocytes to contribute to regulation of plasma heavy-metal ions and may mediate responses to Zn2+ released into the portal circulation with insulin. (+info)
Characterization of the actin binding properties of the vasodilator-stimulated phosphoprotein VASP.
The vasodilator-stimulated phosphoprotein (VASP) colocalizes with the ends of stress fibers in cell-matrix and cell-cell contacts. We report here that bacterially expressed murine VASP directly interacts with skeletal muscle actin in several test systems including cosedimentation, viscometry and polymerization assays. It nucleates actin polymerization and tightly bundles actin filaments. The interaction with actin is salt-sensitive, indicating that the complex formation is primarily based on electrostatic interactions. Actin binding is confined to the C-terminal domain of VASP (EVH2). This domain, when expressed as a fusion protein with EGFP, associates with stress fibers in transiently transfected cells. (+info)
Alteration of iron homeostasis following chronic exposure to manganese in rats.
Recent studies suggest that manganese-induced neurodegenerative toxicity may be partly due to its action on aconitase, which participates in cellular iron regulation and mitochondrial energy production. This study was performed to investigate whether chronic manganese exposure in rats influenced the homeostasis of iron in blood and cerebrospinal fluid (CSF). Groups of 8-10 rats received intraperitoneal injections of MnCl2 at the dose of 6 mg Mn/kg/day or equal volume of saline for 30 days. Concentrations of manganese and iron in plasma and CSF were determined by atomic absorption spectrophotometry. Rats exposed to manganese showed a greatly elevated manganese concentration in both plasma and CSF. The magnitude of increase in CSF manganese (11-fold) was equivalent to that of plasma (10-fold). Chronic manganese exposure resulted in a 32% decrease in plasma iron (p<0.01) and no changes in plasma total iron binding capacity (TIBC). However, it increased CSF iron by 3-fold as compared to the controls (p<0.01). Northern blot analyses of whole brain homogenates revealed a 34% increase in the expression of glutamine synthetase (p<0.05) with unchanged metallothionein-I in manganese-intoxicated rats. When the cultured choroidal epithelial cells derived from rat choroid plexus were incubated with MnCl2 (100 microM) for four days, the expression of transferrin receptor mRNA appeared to exceed by 50% that of control (p<0.002). The results indicate that chronic manganese exposure alters iron homeostasis possibly by expediting unidirectional influx of iron from the systemic circulation to cerebral compartment. The action appears likely to be mediated by manganese-facilitated iron transport at brain barrier systems. (+info)
Improved DNA sequencing accuracy and detection of heterozygous alleles using manganese citrate and different fluorescent dye terminators.
The use of dideoxynucleotide triphosphates labeled with different fluorescent dyes (dye terminators) is the most versatile method for automated DNA sequencing. However, variation in peak heights reduces base-calling accuracy and limits heterozygous allele detection, favoring use of dye-labeled primers for this purpose. We have discovered that the addition of a manganese salt to the PE Applied Biosystems dye-terminator sequencing kits overcomes these limitations for the older rhodamine dyes as well as the more recent dichloro-rhodamine dyes (dRhodamine and BigDyes). Addition of manganese to reactions containing dRhodamine-based dye terminators produced the highest base-calling accuracy. This combination resulted in the most uniform electropherogram profiles, superior to those produced by BigDye terminators and published for dye primers, and facilitated detection of heterozygous alleles. (+info)