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Isolation and purificatiuon of phosphoglycerate kinase from leaves of amaranth ( Amaranthus cruentus, L.)

By: Contributor(s): Material type: TextTextPublication details: Tbilisi (Georgia) CIMMYT : 2004Description: p. 381Subject(s): DDC classification:
  • 633.1147 BED
Summary: Phosphoglycerate kinase (PGK; EC 2.7.2.3) catalyzes the reversible interconversion of 3- phosphoglycearte and 1,3-diphosphoglycearte with the concomitant utilization or generation of ATP. This enzyme participates in reductive pentosephosphate cycle, glycolysis, and glyconeogenesis and is found in all cellular organisms (Shah, Bradbeer, 1994). PGK is located in the soluble fractions of the cytoplasm and chloroplasts of the mesophyll and bundle sheath cells of amaranth leaves (Allahverdiev, Bayramov, Guliev, 1999). Separation from amaranth leaves and purification of PGK was carried out. The studied enzyme precipitated from an extract of leaves at 83% saturation of ammonium sulfate. The pellet was re-suspended in a minimal volume of buffer (40 mM Tris-HCI (pH 8,0), 5 mM MgSO 4 10 mM 2- mercaptoethanol), dialyzed and then loaded onto a DEAE-cellulose column. The enzyme was eluted with a 0 to 300 mM KCI gradient in the same buffer. Further purification of PGK was carried out by a gel filtration on Sephadex G200, equilibrated with the same buffer containing 20 mM NaCI. The assay for the PGK activity was carried out spectrophotometrically by a linked reaction with glyceraldehyde-3-phosphate dehydrogenase (McFadden, Schuster,1972). Protein concentration was estimated by the Lowry method (Lowry, 1954). The molecular mass of native PGK of amaranth leaves was estimated to be 41 kDa on the basis of gel filtration through Sephadex G200. For initiation of PGK activity presence of Mg2+ ions is necessary. PGK of amaranth leaves is relatively thermostable, having a wide pH optimum (7,2-9,8). The dependence of PGK activity on the 3- phosphoglycearte and MgATp2- concentrations does not follow Michaelis-Menten equation, showing nonlinear kinetics. Michaelis constant of PGK for 3-phosphoglycearte was 0.95 mM and 0,33 mM for MgATp2-. High concentrations of MgATp2- inhibited PGK activity. Na2S04 was found to act as the enzyme inhibitor. Anion-exchange chromatography on a DEAE-sephacel ascertained presence of two isoforms of PGK in leaves of amaranth: the first isoform had 88,5% and the second 11,5% of the total activity.
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Conference proceedings CIMMYT Knowledge Center: John Woolston Library CIMMYT Publications Collection 633.1147 BED (Browse shelf(Opens below)) 1 Available 5R630072
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Abstract only

Phosphoglycerate kinase (PGK; EC 2.7.2.3) catalyzes the reversible interconversion of 3- phosphoglycearte and 1,3-diphosphoglycearte with the concomitant utilization or generation of ATP. This enzyme participates in reductive pentosephosphate cycle, glycolysis, and glyconeogenesis and is found in all cellular organisms (Shah, Bradbeer, 1994). PGK is located in the soluble fractions of the cytoplasm and chloroplasts of the mesophyll and bundle sheath cells of amaranth leaves (Allahverdiev, Bayramov, Guliev, 1999). Separation from amaranth leaves and purification of PGK was carried out. The studied enzyme precipitated from an extract of leaves at 83% saturation of ammonium sulfate. The pellet was re-suspended in a minimal volume of buffer (40 mM Tris-HCI (pH 8,0), 5 mM MgSO 4 10 mM 2- mercaptoethanol), dialyzed and then loaded onto a DEAE-cellulose column. The enzyme was eluted with a 0 to 300 mM KCI gradient in the same buffer. Further purification of PGK was carried out by a gel filtration on Sephadex G200, equilibrated with the same buffer containing 20 mM NaCI. The assay for the PGK activity was carried out spectrophotometrically by a linked reaction with glyceraldehyde-3-phosphate dehydrogenase (McFadden, Schuster,1972). Protein concentration was estimated by the Lowry method (Lowry, 1954). The molecular mass of native PGK of amaranth leaves was estimated to be 41 kDa on the basis of gel filtration through Sephadex G200. For initiation of PGK activity presence of Mg2+ ions is necessary. PGK of amaranth leaves is relatively thermostable, having a wide pH optimum (7,2-9,8). The dependence of PGK activity on the 3- phosphoglycearte and MgATp2- concentrations does not follow Michaelis-Menten equation, showing nonlinear kinetics. Michaelis constant of PGK for 3-phosphoglycearte was 0.95 mM and 0,33 mM for MgATp2-. High concentrations of MgATp2- inhibited PGK activity. Na2S04 was found to act as the enzyme inhibitor. Anion-exchange chromatography on a DEAE-sephacel ascertained presence of two isoforms of PGK in leaves of amaranth: the first isoform had 88,5% and the second 11,5% of the total activity.

English

0409|AGRIS 0401|AL-Maize Program

Juan Carlos Mendieta

CIMMYT Publications Collection


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