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Purification and characterization of phosphoglucomutase from peas
Author(s) -
Galloway Cynthia M.,
Dugger W. Mack
Publication year - 1994
Publication title -
physiologia plantarum
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.351
H-Index - 146
eISSN - 1399-3054
pISSN - 0031-9317
DOI - 10.1111/j.1399-3054.1994.tb08839.x
Subject(s) - phosphoglucomutase , pisum , chromatography , molecular mass , enzyme assay , high performance liquid chromatography , chemistry , polyacrylamide gel electrophoresis , enzyme , biochemistry , histidine
Phosphoglucomutase (EC 2.7.5.1) was isolated from pea seeds ( Pisum sativum L. cv. Grenadier) and purified to homogeneity as determined by sodium dodecylsulfate ‐ polyacrylamide gel electrophoresis (SDS‐PAGE) analysis. The enzyme was purified by utilizing 25% polyethylene glycol 4000 precipitation, followed by Fractogel‐diethyla‐minoethyl (DEAE) 650. Fractogel‐TSK HW‐55(s). and high pressure liquid chroma‐tography (HPLC)‐(PEI) column chrornatography. The resulting enzyme had a specific activity of 157 units (mg protein)‐1. a 152‐fold increase over that of the crude plant extract. The molecular weight of the enzyme was 128 to 136 kDa. as determined by native‐PAGE and column chromatography, and when it was subjected to SDS‐PAGE analysis, it was found to be composed of two subunits having molecular weights ranging from 59 to 64 kDa. Upon SDS‐PAGE analysis of a sample purified through HPLC‐PEI chromatography. two bands of protein were found: one having a molecular weight of 64 kDa and the other 68 kDa. A pH optimum of 8.6 was found for the enzyme while it was also found that cysleine. Mg 2+ and glucose 1.6‐bisphosphate were necessary for optimal activity Histidine and imidazole only partially fulfilled the cysteine requirement. A 20‐min preincubation period in the absence of glucose 1‐phosphate was necessary for optimal activity of the enzyme. Without a preincubation period, there was a pronounced lag preceding the linear portion of the reaction as well as a reduction in the V max . An analysis of the kinetics of the reaction showed K m values ot 3.6 × 10 −5 and 1.45 × 10 −5 M for glucose 1‐phosphate and glucose 1.6‐bisphosphate. respectively. A K., of 7.3 × 10 −5 M was obtained for MgCl 2 .