Evidence for a Light Dependent Increase of Phosphoglucomutase Activity in Isolated, Intact Spinach Chloroplasts

Abstract

Phosphoglucomutase (PGM) activity was measured in spinach (Spinacia oleracea L.) chloroplasts. Initial enzyme activity in a chloroplast lysate was 5 to 10% of total activity measured with 1 micromolar glucose 1,6-bisphosphate (Glcl,6-P2) in the assay. Initial PGM activity increased 2-to 3-fold when chloroplasts were illuminated for 10 minutes prior to enzyme measurement and then decreased slowly in the dark. Measurements of total enzyme activity were unchanged by prior light treatment. Inifial PGM activity from light treated chloroplasts was sufficient to account for in vivo rates of starch synthesis. Changes in PGM activity were affected by stromal pH and orthophosphate concentration. Photosynthetic inhibitors, DL-glyceraldehyde, glycolaldehyde, and glyoxylate, decreased and 3-phosphoglyceric acid increased light induced changes of PGM activity. Dark preincubation of chloroplasts with 10 millimolar dithiothreitol had no effect upon initial PGM activity, suggesting that light effects did not involve a sulfhydryl mechanism. Hexose monophosphate levels increased in illuminated chloroplasts. Activation of PGM in a chloroplast lysate by Glcl,6-P2 was maximal between pH 7.5 and 8.5. Stromal concentrations of Glcl,6-P2 were between 20 and 30 micromolar for both light and dark incubated chloroplasts and these levels should saturate PGM activity. Light dependent alterations of enzyme activity may be due to changes of phosphorylated PGM levels in the stroma or are the result of changes in residual activity by the dephosphorylated form of the enzyme. The above results indicate that PGM activity in spinach chloroplasts may be regulated by light, stromal pH, and Glc1,6-P2 concentration.

Photosynthate Metabolism in the Source Leaves of N2-Fixing Soybean Plants

“…Again, the steady-state plastidic glucose-6-P levels were computed to be the highest in the plastids of the N2Fx plants, but PGM may not be a major rate-limiting factor because its total leaf activities are as much as 40 times higher than that of the ADPG-PPiase (Table II). Because we did not measure the soybean leaf chloroplast and cytosolic isoforms of PGM separately (19), the assessment of the role of this enzyme in relationship to carbon-nitrogen interactions remains for considerable future examination (29,31). Recent research by Sicher (29) and Sicher and Kremer (31) has made it clear that the PGM cofactor, glucose-1,6-bisP, is a light activation component in attaining maximal activitiy of PGM during the dark to light transition.…”

“…Preparations of freshly extracted PGM from animal tissues do not require glucose-1,6-bisP as a cofactor, as long as the enzyme remains phosphorylated (9). In contrast, green plant PGM is not phosphorylated in vivo, and requires glucose-1,6-bisP as an obligate cofactor (9,29). We now report that soybean leaf PGM also required glucose-1,6-bisP to support activity, and PGM activities in leaf extracts from all treatments were negligible in the absence of that cofactor (Fig.…”

“…Fructose-2,6-bisP was separated and quantitated employing the methods and modifications described previously (2,30,31). Employing the procedures previously developed (29,31), glucose-1,6-bisP levels were quantitated in a common extract from leaf(lets) at node five. Subsequent to perchlorate extraction and quantitation, the concentration of these compounds were originally expressed on a mg pheophytin basis that subsequently was extrapolated to a mg Chl basis (31).…”

“…Based upon prior studies and upon comparisons of total leaf and chloroplast light-dark transients of glucose-i-P with those of glucose-6-P and fructose-6-P, it was assumed that glucose-i-P would have a potential range of 25 to 55% of the leaf total located in the chloroplast (10,22). Fructose-2,6-bisP was assumed to be entirely in the cytosol (13), and 50% of the glucose-1,6-bisP was assumed to be in the cytosol and the other 50% in the chloroplast (29,31 FBPase: product detected, fructose-6-P; PHI: product detected, glucose-6-P; PGM: product detected; glucose-6-P; and ADPG-PPiase: product detected, glucose-i-P. …”

“…Authentic glucose-1,6-bisP was obtained from Sigma. Reaction mixture conditions for soybean leaf PGM were established experimentally in this laboratory using modifications of previously decribed procedures (9,19,29). Preparations of freshly extracted PGM from animal tissues do not require glucose-1,6-bisP as a cofactor, as long as the enzyme remains phosphorylated (9).…”