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Publisher Summary This chapter describes the assay method and properties of glyceraldehyde-3-phosphate dehydrogenase isolated from spinach leaves. Two different glyceraldehyde-3-phosphate dehydrogenases exist in green leaves. One, localized in the chloroplast, is active with either nicotinamide adenine dinucleotide phosphate kinase (NADP + ) or nicotinamide adenine dinucleotide kinase (NAD + ) and is thought to function in the photosynthetic production of hexoses. The other, located in the cytoplasm, requires NAD+ for activity and is associated with the glycolytic process. The enzyme is assayed spectrophotometrically at 25°C by measuring the rate of increase in absorbance at 340 nm because of the reduction of NAD + in the presence of arsenate. The steps involved in the purification of the enzyme are: (1) extraction, (2) heat fractionation, (3) ammonium sulfate fractionation, (4) acetone fractionation, and (5) affinity chromatography. All operations are carried out at 0–4°C. Ammonium sulfate fractionations are made by the slow addition, under stirring, of solid salt. Centrifugations are carried out at 14,000 g for 30 min. Ultrafiltrations are performed by an Amicon cell equipped with PM-30 membrane. |
