Soybean seed galactinol synthase activity as determined by a novel colorimetric assay

Abstract

-Galactinol synthase (GS) is a key enzyme for the biosynthesis of raffinose oligosaccharides (RO) which are the flatulence factors present in soybean seeds and several other legumes. Understanding of soybean seed GS properties is, therefore, of biotechnological interest. The GS enzyme catalyses formation of galactinol and UDP from UDP-gal and myo-inositol. This enzyme is currently assayed by an isotopic method. We have then idealized a more convenient method for GS assay based on the indirect colorimetric determination of the UDP formed which is then hydrolyzed by exogenous apyrase and the resulting Pi quantified by a modification of the colorimetric method of Fiske & SubbaRow. The color developed is stable, and the method is suitable for detection of very low GS activity.The GS activity profiles of developing soybean seeds determined by the isotopic and the colorimetric methods are closely related. The GS enzyme was partially purified (46-fold) by treatment of seed extract with MnCl 2 , sequential chromatographies on DEAE-Sepharose, Phenyl-Sepharose CL-4B and Q-Sepharose columns. The crude and the partially purified enzyme showed maximum activity at pH 7.0 and 50 ºC. Dithiothreitol and MnCl 2 enhanced considerably the activity of the partially purified enzyme. While UDP-glc could be hydrolyzed by the enzyme at a reative activity corresponding to 49% of that calculated for UDP-gal, UDP-man and sucrose were completely ineffective as alternative substrates. ADDITIONAL INDEX TERMS:Flatulence, galactinol synthase, colorimetric assay, raffinose oligosaccharides. ABBREVIATIONS -GS ATIVIDADE DE GALACTINOL SINTASE DE SEMENTE DE SOJA DETERMINADA POR UM NOVO ENSAIO CALORIMÉTRICORESUMO -Galactinol sintase (GS) é a enzima-chave para a biossíntese de oligosacarídeos de rafinose (RO), que são os fatores antinutricionais causadores de flatulência, os quais estão presentes em sementes de soja e em outros legumes. A GS catalisa a formação de galactinol e UDP a partir de UDP-gal e A atividade dessa enzima é determinada atualmente pelo método radioisotópico que, apesar de adequado tecnicamente, apresenta vários inconvenientes, tais como a necessidade de substrato de alto custo, bem como de cuidados adicionais e serviços especializados para descarte dos resíduos radioativos. Assim, desenvolveu-se um método colorimétrico alternativo ao método radioisotópico, baseado na determinação colorimétrica indireta do UDP formado pela hidrólise enzimática (apirase) desse nucleotídeo e determinação do Pi resultante pelo método de Fiske & SubbaRow, com modificações. A cor desenvolvida é estável e o método é sensível para detecção de quantidades nanomolares de Pi.Os perfis de atividade da GS em sementes de soja em diferentes fases de desenvolvimento, determinados pelos métodos colorimétrico e radioisotópico, são semelhantes. Adicionalmente, a GS de sementes de soja foi purificada (46-vezes) por tratamento do extrato das sementes com MnCl 2 , e uma seqüência de cromatografias em colunas de DEAE-Sepharose, Phenyl-Sepharose CL-4B e Q-Sepha...

VvGOLS1 and VvHsfA2 are Involved in the Heat Stress Responses in Grapevine Berries

“…Crude protein extracts from empty vector control cultures lacked this synthetic capacity. The optimal pH, apparent K m and V max for the two GOLS substrates determined for recombinant VvGOLS1 were similar to those reported from other plants (Bachmann et al 1994, Liu et al 1995, Ribeiro et al 2000, Wakiuchi et al 2003, Li et al 2011. The mechanisms involved in the transcriptional regulation of GOLS genes are poorly known.…”

Isolation and characterization of galactinol synthases from hybrid poplar

“…Relative expression (%) was determined colorimetrically (Ribeiro et al ., 2000). Error bars correspond to the standard error of the mean.…”

Responses of Populus trichocarpa galactinol synthase genes to abiotic stresses

“…5 ), which suggests the involvement of other PtrGolS genes in salt- and water-deficit responses, particularly early responses. Many studies have reported that GolS isozymes differ in their enzymatic properties as well as physiological conditions for their activities (Bachmann et al 1994 ; Liu et al 1995 ; Riberio et al 2000 ; Unda et al 2012 ). Further characterization of PtrGolS proteins is thus required to elucidate the roles of GolS in RFO biosynthesis in response to abiotic stresses.…”

“…Galactinol is synthesized from UDP-D-galactose and myo -inositol by galactinol synthase (GolS; EC 2.4.1.123) (Liu et al 1995 ; Saravitz et al 1987 ). Extensive biochemical studies have characterized GolS in many plant species, including the common bugle ( Ajuga reptans ) (Bachmann et al 1994 ), zucchini squash ( Cucurbita pepo ) (Liu et al 1995 ; Smith et al 1991 ), kidney bean ( Phaseolus vulgaris ) (Liu et al 1995 ), soybean ( Glycine max ) (Riberio et al 2000 ) and cucumber ( Cucumis sativus ) (Wakiuchi et al 2003 ). In addition, molecular biological approaches have accelerated the studies of stress responses of GolS genes.…”