Partial purification and characterization of phosphoglucomutase from developing grains of thermotolerant wheat (Triticum aestivum L.em.Thell)
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Date
2012
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Publisher
CCSHAU
Abstract
Wheat grain is the dominant grain of world commerce and is the staple food of millions of
people world wide. High temperature beyond 30 0C, which is usually encountered during later part of
grain filling period, affects grain yield (reduction by 20-50 per cent) and grain quality. Starch is the
major storage carbohydrate in wheat grains. It is synthesized from sucrose, which is the principal
product of leaf photosynthesis and transported to the wheat grain. Phosphoglucomutase (PGM)is an
important enzyme in the conversion of sucrose to starch in the developing wheat grains, as it facilitates
the interconversion of glucose-1-phosphate and glucose-6-phosphate.The PGM reaction is thought to
be important in the partitioning of carbon between the pathways of starch synthesis and carbohydrate
oxidation. Keeping above in view, the present investigations were conducted to purify and characterize
phosphoglucomutase from developing grains of thermotolerant wheat. Phosphoglucomutase was
purified to near homogeneity (as revealed by single band on Native-PAGE) from immature grains (21
days after anthesis) of thermotolerant wheat WH-1021 by using conventional protein purification
techniques viz. 40-60% ammonium sulphate fractionation, gel filtration through sephadex G-100 and
DEAE-cellulose ion exchange chromatography. The enzyme was purified about 22 fold with 28.67 per
cent recovery. The molecular weight as determined by gel filtration and subunit molecular weight as
determined by SDS-PAGE (single band) were found to be 130 kDa and 66 kDa, respectively indicating
that enzyme is a homodimer. The purified enzyme exhibited optimum activity at pH 7.5 and 350C. It
was thermostable upto 550C. The enzyme followed Michaelis-Menten kinetics with Km value of 0.8
mM for G-1-P as a substrate. Amongst various metabolites tested AMP, ADP, NADPH and F-2,6-BP
were found to be the potent inhibitors of purified phosphoglucomutase, inhibiting the enzyme activity
by 26, 12, 9 and 23%, respectively.The enzyme activity was stimulated by Mn2+, Mg2+ and SO4
2- while
Na+ and Ca2+ inhibited the activity at 2 mM concentration.To summarize, higher thermostability of
enzyme is suggestive of enzyme’s adaptation to high temperature stress.
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Keywords
Phosphoglucomutase, Triticum aestivum, Starch