Phosphoglucomutase (EC 2.7.5.1) was isolated from pea seeds (Pisum sat
ivum L. cv. Grenadier) and purified to homogeneity as determined by so
dium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) anal
ysis. The enzyme was purified by utilizing 25% polyethylene glycol 400
0 precipitation, followed by Fractogel-diethylaminoethyl (DEAE) 650, F
ractogel-TSK HW-55(s), and high pressure liquid chromatography (HPLC)-
(PEI) column chromatography. The resulting enzyme had a specific activ
ity 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 chromatograph
y, two bands of protein were found; one having a molecular weight of 6
4 kDa and the other 68 kDa. A pH optimum of 8.6 was found for the enzy
me while it was also found that cysteine, Mg2+ and glucose 1,6-bisphos
phate were necessary for optimal activity. Histidine and imidazole onl
y partially fulfilled the cysteine requirement. A 20-min preincubation
period in the absence of glucose 1-phosphate was necessary for optima
l 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 Km values of 3.6 x 10(-5) and 1.45 x 10(-7) M for glucose 1-ph
osphate and glucose 1,6-bisphosphate, respectively. A K-a of 7.3 x 10(
-5) M was obtained for MgCl2.