ABSTRACT
Four putative apyrase genes were identified from the model legume Medicago truncatula. Two of the genes identified from M. truncatula (Mtapy1 and Mtapy4) are expressed in roots and are inducible within 3 h after inoculation with Sinorhizobium meliloti. The level of mRNA expression of the other two putative apyrases, Mtapy2 and Mtapy3, was unaffected by rhizobial inoculation. Screening of a bacterial artificial chromosome library of M. truncatula genomic DNA showed that Mtapy1, Mtapy3, and Mtapy4 are present on a single bacterial artificial chromosome clone. This apyrase cluster was mapped to linkage group seven. A syntenic region on soybean linkage group J was found to contain at least two apyrase genes. Screening of nodulation deficient mutants of M. truncatula revealed that two such mutants do not express apyrases to any detectable level. The data suggest a role for apyrases early in the nodulation response before the involvement of root cortical cell division leading to the nodule structure.
Subject(s)
Apyrase/genetics , Gene Expression Regulation, Plant , Medicago sativa/enzymology , Medicago sativa/genetics , Sinorhizobium/physiology , Transcription, Genetic , Amino Acid Sequence , Apyrase/biosynthesis , Apyrase/chemistry , Conserved Sequence , Enzyme Induction , Gene Expression Regulation, Enzymologic , Humans , Isoenzymes/biosynthesis , Isoenzymes/chemistry , Isoenzymes/genetics , Medicago sativa/microbiology , Molecular Sequence Data , Plant Roots/enzymology , Plants/enzymology , Plants/genetics , Polymerase Chain Reaction , RNA, Messenger/genetics , Sequence Alignment , Sequence Homology, Amino Acid , Time FactorsABSTRACT
The N400 component of event-related potentials (ERPs) was obtained in a modified version of the Neely [J.H. Neely, Semantic priming and retrieval from lexical memory: Roles of inhibitionless spreading activation and limited-capacity attention, Journal of Experimental Psychology: General, Vol. 106 (1977), pp. 226-254.] paradigm which permits unconfounding of semantic priming effects due to automatic and attentional processes. It was found that a short stimulus onset asynchrony (SOA) of 250 ms between the prime and the target was associated with automatic but not expectancy effects on the amplitude of N400. At a long SOA of 2000 ms between prime and target, semantic priming effects on N400 were obtained associated with both automatic and expectancy processes. Moreover, there was no significance difference in the magnitude of the automatic effects at the two SOAs, suggesting that automatic processing had not decayed within the 2000 ms interval between the prime and target. The results support the two-processing interpretation of semantic priming advanced by Posner and Synder [M.I. Posner, C.R.R. Snyder, Attention and cognitive control, in: R.L. Solso (Ed.), Information Processing and Cognition: The Loyola Symposium, Erlbaum, Hillsdale, NJ (1975).] and concur with the results of Neely [J.H. Neely, Semantic priming and retrieval from lexical memory: Roles of inhibitionless spreading activation and limited-capacity attention, Journal of Experimental Psychology: General, Vol. 106 (1977), pp. 226-254], with the exception of indicating a longer persistence of automatic processes.
Subject(s)
Brain/physiology , Evoked Potentials/physiology , Language , Memory/physiology , Reaction Time , Adult , Electroencephalography , Female , Humans , Linguistics , Male , Reading , SemanticsABSTRACT
Endoinulinase from Aspergillus ficuum, which catalyzes the hydrolysis of inulin via an endo-cleavage mode, was purified by chromatography from Novozym 230 as a starting commercial enzyme mixture on CM-Sephadex and DEAE-Sepharose, and by preparative electrophoresis under native conditions. The enzyme was estimated to be pure on the basis of its I/S ratio, whose value was infinite in our assay conditions. Two forms separated by using this method. SDS gel electrophoresis showed the two purified forms to respectively exhibit molecular weights of 64,000 +/- 500 and 66,000 +/- 1,000. The results of deglycosylation indicated that the two forms were originally the same protein but with different sugar contents. A molecular weight of 54,800 +/- 1,500 was found by gel filtration of the native enzyme, indicating the native functional protein to be a monomer. The enzyme showed nearly absolute substrate specificity towards inulin and inulooligosaccharides, and acted via an endo-attack to produce mainly inulotriose during the late stage of the reaction. The apparent Km and Vmax values for inulin hydrolysis were 8.1 +/- 1.0 mM and 773 +/- 60 U/mg, respectively. The internal peptides of the enzyme showed sequence homology to the endoinulinase of Penicillium purpurogenum.
Subject(s)
Aspergillus/enzymology , Glycoside Hydrolases/isolation & purification , Amino Acid Sequence , Aspergillus/genetics , Glycoside Hydrolases/genetics , Glycoside Hydrolases/metabolism , Hydrogen-Ion Concentration , Inulin , Kinetics , Metals , Molecular Sequence Data , Molecular Weight , Peptide Fragments/genetics , Peptide Fragments/isolation & purification , Substrate Specificity , TemperatureABSTRACT
Thiol-specific antioxidant (TSA) from yeast contains cysteine residues at amino acid positions 47 and 170 but is not associated with obvious redox cofactors. These two cysteines are highly conserved in a family of proteins that exhibit sequence identity of 23-98% with TSA. The roles of Cys-47 and Cys-170 in yeast TSA were investigated by replacing them individually with serine and expressing the mutant TSA proteins (RC47S and RC170S, respectively), as well as wild-type TSA (RWT), in Escherichia coli. Wild-type TSA purified from yeast (YWT) and RWT were both shown to exist predominantly as dimers, whereas RC47S and RC170S existed mainly as monomers under a denaturing condition. This observation suggests that the dimerization of YWT and RWT requires disulfide linkage of Cys-47 and Cys-170. The presence of the Cys-47-Cys-170 linkage in YWT was directly shown by isolation of dimeric tryptic peptides, one monomer of which contained Cys-47 and the other contained Cys-170. A small percentage of YWT, RWT, RC47S, and RC170S molecules formed dimers linked by Cys-47-Cys-47 or Cys-170-Cys-170 disulfide bonds. The antioxidant activity of the various TSA proteins was evaluated from their ability to protect glutamine synthetase against the dithiothreitol/Fe3+/O2 oxidation system. YWT, RWT, and RC170S were equally protective, whereas RC47S was completely ineffective. Thus, Cys-47, but not Cys-170, constitutes the site of oxidation by putative substrate.
Subject(s)
Antioxidants/chemistry , Cysteine/chemistry , Peroxidases , Proteins/chemistry , Saccharomyces cerevisiae/chemistry , Amino Acid Sequence , Base Sequence , Biopolymers , Cloning, Molecular , Conserved Sequence , DNA, Fungal , Disulfides , Escherichia coli , Immunoblotting , Molecular Sequence Data , Mutagenesis, Site-Directed , Oxidation-Reduction , PeroxiredoxinsABSTRACT
Thiol-specific antioxidant protein (Protector Protein; PRP) from Saccharomyces cerevisiae was found to remove hydrogen peroxide and hydroxyl radical in the presence of dithiothreitol (DTT). Without DTT as a reducing equivalent, the antioxidant protein did not show the activities for destroying hydrogen peroxide and hydroxyl radical. N-ethylmaleimide (NEM) was observed to prevent the PRP from both removing hydrogen peroxide and protecting the cleavage of DNA. These observations suggest that the sulfhydryl of cysteine in PRP could function as a strong nucleophile to attack and destroy H2O2 and .OH.
