Partial purification and characterization of lysine-ketoglutarate reductase in normal and opaque-2 maize endosperms

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dc.contributor.authorBrochetto-Braga, Marcia R. [UNESP]
dc.contributor.authorLeite, Adilson
dc.contributor.authorArruda, Paulo
dc.contributor.institutionUniversidade Estadual de Campinas (UNICAMP)
dc.contributor.institutionUniversidade Estadual Paulista (Unesp)
dc.date.accessioned2014-05-27T11:17:30Z
dc.date.available2014-05-27T11:17:30Z
dc.date.issued1992-12-01
dc.description.abstractLysine-ketoglutaratc reductase catalyzes the first step of lysine catabolism in maize (Zea mays L.) endosperm. The enzyme condenses L-lysine and α-ketoglutarate into saccharopine using NADPH as cofactor. It is endosperm-specific and has a temporal pattern of activity, increasing with the onset of kernel development, reaching a peak 20 to 25 days after pollination, and thereafter decreasing as the kernel approaches maturity. The enzyme was extracted from the developing maize endosperm and partially purified by ammonium-sulfate precipitation, anion-exchange chromatography on DEAE-cellulose, and affinity chromatography on Blue-Sepharose CL-6B. The preparation obtained from affinity chromatography was enriched 275-fold and had a specific activity of 411 nanomoles per minute per milligram protein. The native and denaturated enzyme is a 140 kilodalton protein as determined by polyacrylamide gel electrophoresis. The enzyme showed specificity for its substrates and was not inhibited by either aminoethyl-cysteine or glutamate. Steady-state product-inhibition studies revealed that saccharopine was a noncompetitive inhibitor with respect to α-ketoglutarate and a competitive inhibitor with respect to lysine. This is suggestive of a rapid equilibriumordered binding mechanism with a binding order of lysine, α-ketoglutarate, NADPH. The enzyme activity was investigated in two maize inbred lines with homozygous normal and opaque-2 endosperms. The pattern of lysine-ketoglutarate reductase activity is coordinated with the rate of zein accumulation during endosperm development. A coordinated regulation of enzyme activity and zein accumulation was observed in the opaque-2 endosperm as the activity and zein levels were two to three times lower than in the normal endosperm. Enzyme extracted from L1038 normal and opaque-2 20 days after pollination was partially purified by DEAE-cellulose chromatography. Both genotypes showed a similar elution pattern with a single activity peak eluted at approximately 0.2 molar KCL. The molecular weight and physical properties of the normal and opaque-2 enzymes were essentially the same. We suggest that the Opaque-2 gene, which is a transactivator of the 22 kilodalton zein genes, may be involved in the regulation of the lysine-ketoglutarate reductase gene in maize endosperm. In addition, the decreased reductase activity caused by the opaque-2 mutation may explain, at least in part, the elevated concentration of lysine found in the opaque-2 endosperm.en
dc.description.affiliationDepartamento de Genética Instituto de Biologia Universidade Estadual de Campinas, 13081 Campinas, SP
dc.description.affiliationCtro. Biol. Molec./Engenharia G. Universidade Estadual de Campinas, 13081 Campinas, SP
dc.description.affiliationDepto Biologia Instituto de Biociéncias UNESP/Rio Claro, Av. 24A, no 1515, 13500 Rio Claro, SP
dc.description.affiliationUnespDepto Biologia Instituto de Biociéncias UNESP/Rio Claro, Av. 24A, no 1515, 13500 Rio Claro, SP
dc.format.extent1139-1147
dc.identifierhttp://www.plantphysiol.org/content/98/3/1139
dc.identifier.citationPlant Physiology, v. 98, n. 3, p. 1139-1147, 1992.
dc.identifier.file2-s2.0-0000690359.pdf
dc.identifier.issn0032-0889
dc.identifier.scopus2-s2.0-0000690359
dc.identifier.urihttp://hdl.handle.net/11449/64316
dc.language.isoeng
dc.relation.ispartofPlant Physiology
dc.relation.ispartofjcr5.949
dc.relation.ispartofsjr3,690
dc.rights.accessRightsAcesso restrito
dc.sourceScopus
dc.titlePartial purification and characterization of lysine-ketoglutarate reductase in normal and opaque-2 maize endospermsen
dc.typeArtigo
dcterms.licensehttp://www.plantcell.org/site/misc/ifora.xhtml
unesp.campusUniversidade Estadual Paulista (Unesp), Instituto de Biociências, Rio Claropt

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Artigos - Biologia - IBRC