Characterization of the Kinetic, Regulatory, and Structural Properties of ADP-glucose Pyrophosphorylase from Chlamydomonas reinhardtii

A.A. IGLESIAS, Y-Y CHARNG, S. BALL, J. PREISS

PLANT PHYSIOLOGY.

Año: 1994 vol. 104 p. 1287 - 1294

0032-0889

ADP-glucose pyrophosphorylase (ADP-Clc PPase) from Chlamydomonasreinhardtii cells was purified over 2000-fold to a specificactivity of 81 units/mg protein, and its kinetic and regulatoryproperties were characterized. lnorganic orthophosphate and 3-phosphoglycerate were the most potent inhibitor and activator,respectively. Rabbit antiserum raised against the spinach leaf ADPClcPPase (but not the one raised against the enzyme from Escherichiacoli) inhibited the activity of the purified algal enzyme, whichmigrated as a single protein band in native polyacrylamide gelelectrophoresis. Two-dimensional and sodium dodecyl sulfatepolyacrylamidegel electrophoresis indicate that the enzyme fromC. reinhardtii is composed of two subunits with molecular massesof 50 and 53 kD, respectively. lhe molecular mas of the nativeenzyme is estimated to be 210 kD. Antisera raised against thespinach leaf holoenzyme and against the 51-kD spinach subunitcross-reacted with both subunits of the algal ADP-Clc PPase inimmunoblot hybridization, but the cross-reaction was stronger forthe 50-kD algal subunit than for the 53-kD subunit. No crossreactionwas observed when antiserum raised against the spinachleaf pyrophosphorylase 54-kD subunit was used. These resultssuggest that the ADP-Clc PPase from C. reinhardtii is a heterotetramericprotein, since the enzyme from higher plants and its twosubunits are structurally more related to the small subunit of thespinach leaf enzyme than to its large subunit. This information isdiscussed in the context of the possible evolutionary changesleading from the bacterial ADP-Clc PPase to the cyanobacterialand higher plant enzymes.