The inactivation of saccharopine dehydrogenase (L-lysine-forming) by diethyl pyrocarbonate.

AbstractThe brown planthopper Nilaparvata lugens is a serious phloem-feeding pest of rice in China. The current study focuses on a saccharopine dehydrogenase (SDH) that catalyzes the penultimate reaction in biosynthesis of the amino acid lysine (Lys), which plays a role in insect growth and carnitine production (as a substrate). The protein, provisionally designated as NlylsSDH [a SDH derived from yeast-like symbiont (YLS) in N. lugens], had a higher transcript level in abdomens, compared with heads, wings, legs and thoraces, which agrees with YLS distribution in N. lugens. Ingestion of Nlylssdh targeted double-stranded RNA (dsNlylssdh) for 5, 10 and 15 days decreased the mRNA abundance in the hoppers by 47, 70 and 31%, respectively, comparing with those ingesting normal or dsegfp diets. Nlylssdh knockdown slightly decreased the body weights, significantly delayed the development of females, and killed approximately 30% of the nymphs. Moreover, some surviving adults showed two apparent phenotypic defects: wing deformation and nymphal cuticles remained on tips of the legs and abdomens. The brachypterours/macropterours and sex ratios (female/male) of the adults on the dsRNA diet were lowered compared with the adults on diets without dsRNA. These results suggest that Nlylssdh encodes a functional SDH protein. The adverse effect of Nlylssdh knockdown on N. lugens implies the importance of Lys in hopper development. This study provides a proof of concept example that Nlylssdh could serve as a possible dsRNA-based pesticide for planthopper control.

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The catabolic function of the α-aminoadipic acid pathway in plants is associated with unidirectional activity of lysine–oxoglutarate reductase, but not saccharopine dehydrogenase

Biochemical Journal ◽

10.1042/bj3510215 ◽

2000 ◽

Vol 351 (1) ◽

pp. 215-220

Author(s):

Xiaohong ZHU ◽

Guiliang TANG ◽

Gad GALILI

Keyword(s):

Adipic Acid ◽

Expression System ◽

Yeast Cells ◽

Deletion Mutants ◽

Null Mutant ◽

Anabolic Activity ◽

Saccharopine Dehydrogenase ◽

Intermediate Metabolites ◽

Acid Pathway ◽

Transcriptional Controls

Whereas plants and animals use the α-aminoadipic acid pathway to catabolize lysine, yeast and fungi use the very same pathway to synthesize lysine. These two groups of organisms also possess structurally distinct forms of two enzymes in this pathway, namely lysine–oxoglutarate reductase (lysine–ketoglutarate reductase; LKR) and saccharopine dehydrogenase (SDH): in plants and animals these enzymes are linked on to a single bifunctional polypeptide, while in yeast and fungi they exist as separate entities. In addition, yeast LKR and SDH possess bi-directional activities, and their anabolic function is regulated by complex transcriptional and post-transcriptional controls, which apparently ascertain differential accumulation of intermediate metabolites; in plants, the regulation of the catabolic function of these two enzymes is not known. To elucidate the regulation of the catabolic function of plant bifunctional LKR/SDH enzymes, we have used yeast as an expression system to test whether a plant LKR/SDH also possesses bi-directional LKR and SDH activities, similar to the yeast enzymes. The Arabidopsis enzyme complemented a yeast SDH, but not LKR, null mutant. Identical results were obtained when deletion mutants encoding only the LKR or SDH domains of this bifunctional polypeptide were expressed individually in the yeast cells. Moreover, activity assays showed that the Arabidopsis LKR possessed catabolic, but not anabolic, activity, and its uni-directional activity stems from its structure rather than its linkage to SDH. Our results suggest that the uni-directional activity of LKR plays an important role in regulating the catabolic function of the α-amino adipic acid pathway in plants.

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Chemical modification of contractile 3-nm-diameter filaments in Vorticella spasmoneme by diethyl-pyrocarbonate and its reversible renaturation by hydroxylamine

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2003.09.121 ◽

2003 ◽

Vol 310 (4) ◽

pp. 1067-1072 ◽

Cited By ~ 6

Author(s):

Jie Fang ◽

Bei Zhang ◽

Hiroshi Asai

Keyword(s):

Chemical Modification ◽

Diethyl Pyrocarbonate

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A simle method for extraction of RNA from E. coli utilizing diethyl pyrocarbonate

Analytical Biochemistry ◽

10.1016/0003-2697(70)90316-7 ◽

1970 ◽

Vol 33 (2) ◽

pp. 460-463 ◽

Cited By ~ 128

Author(s):

William C. Summers

Keyword(s):

E Coli ◽

Diethyl Pyrocarbonate

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Genetic control of lysine metabolism in maize endosperm mutants

Functional Plant Biology ◽

10.1071/fp03173 ◽

2004 ◽

Vol 31 (4) ◽

pp. 339 ◽

Cited By ~ 31

Author(s):

Ricardo A. Azevedo ◽

Catherine Damerval ◽

Peter J. Lea ◽

Jacques Landry ◽

Cláudia M. Bellato ◽

...

Keyword(s):

Amino Acids ◽

Qualitative Study ◽

Aspartate Kinase ◽

Maize Endosperm ◽

Homoserine Dehydrogenase ◽

Saccharopine Dehydrogenase ◽

Endosperm Mutants ◽

Mutant Lines ◽

Mutant Genes ◽

Lysine Metabolism

The capacity of three maize endosperm opaque mutants (o10, o11 and o13) to accumulate soluble lysine in the seed in relation to their wildtype counterpart, W22+, was investigated. The W22o13 and W22o11 mutants exhibited 278% and 186% increases in soluble lysine, respectively, while for W22o10, a 36% decrease was observed, compared with the wildtype. A quantitative and qualitative study of the N constituents of the endosperm has been conducted and data obtained for the total protein, non-protein N, soluble amino acids, albumins / globulins, zeins and glutelins present in the seed of the mutants. Following 2D–PAGE, a total of 38 different forms of zein polypeptides were detected and considerable differences were noted between the three mutant lines. The metabolism of lysine was also studied by analysis of the enzymes aspartate kinase, homoserine dehydrogenase, lysine 2-oxoglutarate reductase and saccharopine dehydrogenase, which exhibited major changes in activity, depending on the genotype, suggesting that the mutant genes may have distinct regulatory activities.

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Short-term toxicity studies on diethyl pyrocarbonate-treated beverages in the rat

Food and Cosmetics Toxicology ◽

10.1016/s0015-6264(72)80001-4 ◽

1972 ◽

Vol 10 (6) ◽

pp. 743-756 ◽

Cited By ~ 1

Author(s):

M. Sharratt ◽

I.F. Gaunt ◽

P. Grasso ◽

Ida S. Kiss ◽

Jean Hooson ◽

...

Keyword(s):

Short Term ◽

Diethyl Pyrocarbonate ◽

Toxicity Studies

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Footprinting studies on the effect of echinomycin on the structure of a bent DNA fragment

Biochemical Journal ◽

10.1042/bj2690217 ◽

1990 ◽

Vol 269 (1) ◽

pp. 217-221 ◽

Cited By ~ 6

Author(s):

K R Fox ◽

E Kentebe

Keyword(s):

Binding Sites ◽

Dna Structure ◽

Drug Binding ◽

Micrococcal Nuclease ◽

Kinetoplast Dna ◽

Bent Dna ◽

Diethyl Pyrocarbonate ◽

Drug Binding Sites ◽

Dna Fragment

The interaction of echinomycin with a kinetoplast DNA fragment which contains phased runs of adenine residues has been examined by various footprinting techniques. DNAase I footprinting confirms that all drug-binding sites contain the dinucleotide CpG. However, not all such sequences are protected. Three sites, each of which is located between two adenine tracks in the sequence GCGA, are not protected from DNAase I attack. Enhanced cleavage by DNAase I, DNAase II and micrococcal nuclease is observed in regions surrounding drug-binding sites. The results suggest that echinomycin alters the conformation of the AT tracks, making them more like an average DNA structure. Echinomycin renders adenine residues in the sequence CGA hyper-reactive to diethyl pyrocarbonate.

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