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ADPglucose pyrophosphorylase has been extensively purified from potato (Solanum tuberosum L.) tuber tissue to study its structure. By employing a modified published procedure (JR Sowokinos, J Preiss [1982] Plant Physiol 69: 1459-1466) together with Mono 0 chromatography, a near homogeneous enzyme preparation was obtained with substantial improvement in enzyme yield and specific activity. In single dimensional sodium dodecyl sulfate polyacrylamide gels, the enzyme migrated as a single polypeptide band with a mobility of about 50,000 daltons. Analysis by two-dimensional polyacrylamide gel electrophoresis, however, revealed the presence of two types of subunits which could be distinguished by their slight differences in net charge and molecular weight. The smaller potato tuber subunit was recognized by antiserum prepared against the smaller spinach leaf 51 kilodalton ADPglucose pyrophosphorylase subunit. In contrast, the anti-54 kilodalton raised against the spinach leaf subunit did not significantly react to the tuber enzyme subunits. The results are consistent with the hypothesis that the potato tuber ADPglucose pyrophosphorylase is not composed of a simple homotetramer as previously suggested, but is a product of two separate and distinct subunits as observed for the spinach leaf and maize enzymes.ADPglucose pyrophosphorylase (ATP: a-glucose-1-P adenylyl-transferase, EC 2.7.7.27) catalyzes an important regulatory step in the biosynthesis of starch and glycogen in plants and bacteria, respectively (2, 16-20). This enzyme mediates the synthesis of ADPglucose and PPi from ATP and glucose-I-P; the product, ADPglucose, serving as the glucosyl donor in a-glucan synthesis. Both the plant and bacterial enzymes are subject to allosteric regulation by small effector molecules 'Supported in part by grants from the

~l h e enzyme UDP-glucose pyrophosphorylase (UCPase) from potato (Solanum tuberosum L. cv Norchip) tubers was purified 177-fold to near homogeneity and to a specific activity of 1099 international unitslmg of protein. l h e molecular mass of the purified enzyme was 53 k D as determined by SDS-PACE and gel filtration. lmmunological and activity assays detected UGPase at similar levels in potato stems, stolons, and tubers. Leaves and roots contained lower levels of UCPase activity and protein. LineweaverBurk plots for substrates inorganic pyrophosphate and UDP-glucose were linear in the pyrophosphorolytic diredion, yielding K,,, values of 0.13 and 0.14 mM, respectively. However, LineweaverBurk plots for the substrates glucose-1-P and UTP were biphasic in nature when UCPase was assayed in the direction of UDP-glucose synthesis. At physiological substrate concentrations (Le. from 0.05-0.20 mM), K,,, values of 0.08 mM (glucose-1-P) and 0.12 mM (UTP) were obtained. When substrate concentrations increased above 0.20 mM, K,,, values increased to 0.68 mM (glucose-1-P) and 0.53

ADPglucose pyrophosphorylase from potato (Solanum tuberosum L.) tubers has been purified by hydrophobic chromatography on 3 aminopropylsepharose (Seph-C3-NH2). The purified preparation showed two closely associated protein-staining bands that coincided with enzyme activity stains. Only one major protein staining band was observed in sodium dodecyl sulfate polyacrylamide gel electrophoresis. The subunit molecular weight was determined to be 50,000. The molecular weight of the native enzyme was determined to be 200,000. The enzyme appeared to be a tetramer consisting of subunits of the same molecular weight. The subunit molecular weight of the enzyme is compared with previously reported subunit molecular weights of ADPglucose pyrophosphorylases from spinach leaf, maize endosperm, and various bacteria. ADPglucose synthesis from ATP and glucose 1-P is almost completely dependent on the presence of 3-P-glycerate and is inhibited by inorganic phosphate. The kinetic constants for the substrates and Mg2+ are reported. The enzyme V,,. is stimulated about 1.5-to 3-fold by 3 millimolar DTT. The significance of the activation by 3-P-glycerate and inhibition by inorganic phosphate ADPglucose synthesis catalyzed by the potato tuber enzyme is discussed.The enzyme ADPglucose pyrophosphorylase (ATP:ca-glucose-I-P adenylyltransferase, E.C. 2.7.7.27), is one of the main regulatory steps in the biosynthesis of a-glucans in bacteria and plants (21,22). This enzyme catalyzes the reversible reaction seen below:ATP + glucose-l-P , ADPglucose + PPi Its regulatory and catalytic properties from several sources have been recently reviewed (22). ADPglucose pyrophosphorylases from leaves of higher plants (23, 24) and nonchlorophyllous reserve tissues (4, 28) are allosterically activated and inhibited by 3-P-glycerate and Pi, respectively. The extensively studied enzyme in maize endosperm (4, 10) is less sensitive to these effectors than those studied in chlorophyllous leaf tissues (24). However, the '

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Changes in ADP-glucose and UDP-glucose pyrophosphorylase activities were followed during tuber development of Solanum tuberosum and prolonged storage at 4 and 11 C. Potato tuberization was accompanied by a sharp increase in starch synthesis simultaneous with a marked rise in ADP-glucose pyrophosphorylase activity. When tubers reached an average diameter of 1 centimeter (0.5 gram average tuber weight) and had already established 58% starch on a dry weight basis, ADP-glucose pyrophosphorylase increased 16-to 24-fold over its activity seen in low starch containing stolon tissue. During this same period UDP-glucose pyrophosphorylase increased approximately 2-to 3-fold. Although participation of UDP-glucose in starch formation can not be neglected, it is suggested that the onset of rapid non-photosynthetic potato tuber starch biosynthesis may be closely related to the simultaneous increase in ADP-glucose pyrophosphorylase activity.Evidence that UDP-glucose and ADP-glucose pyrophosphorylases are separate protein entities was indicated by their (a) activity ratio variations during tuber development and storage, (b) extraction stabilities, (c) morphological localization, (d) separation with ammonium sulfate, (e) pH optima, and (f) differential activation with 3-P-glycerate.