The Subunit Structure of Potato Tuber ADPglucose Pyrophosphorylase

Abstract

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

Multiple Forms of ADP-Glucose Pyrophosphorylase from Tomato Fruit

“…1, lower part of lanes 9 and 10). These results, in agreement with previous studies by Okita et al (1990) and Kleczkowski et al (1993), indicate that the protease activity is also a hurdle to AGP purification from tomato fruit. The presence of protease inhibitors in the grinding solution and extraction and purification at 0 to 4°C were two effective measures in inhibiting the protease activity.…”

“…A typical purification of AGP from tomato fruit is summarized in Table I. By using a procedure consisting of heat treatment, ammonium sulfate precipitation, Affi-Gel blue gel, Q-Sepharose, and Mono-Q chromatography, we were able to purify the enzyme 5018-fold, with 31% recovery, to a final specific activity of about 45 unitsl mg, a value comparable to that reported for the enzyme purified from potato tuber (56.9 units/mg; Okita et al, 1990). However, the purification and yield values are an overestimation because the heat treatment caused a 3.5-fold increase in total enzyme activity (Table I).…”

“…In the case of barley endosperm AGP, the small subunit was relatively resistant to proteolytic degradation but the large subunit was not, with a half-time for proteolysis at 0 to 4°C on the order of minutes, even in the presence of various protease inhibitors (Kleczkowski et al, 1993). In the purification of AGP from potato tuber, low recovery and instability of the enzyme, probably resulting from protease activity, were reported (Okita et al, 1990). Therefore, an E. coli mutant expressing the potato tuber AGP was used as an alternative for the enzyme purification (Iglesias et al, 1993).…”

“…Comparison of cDNA sequences and immunological analysis revealed that small subunits from different sources are highly conserved, whereas the large subunits are divergent (Preiss et al, 1989;Okita et al, 1990;Smith-White and Preiss, 1992). Expression of both subunit cDNAs from potato tubers in Escherichia coli suggests that a major function of the large subunit is to modulate the regulatory properties of the native heterotetrameric enzyme, whereas that of the small subunit is catalysis (Ballicora et al, 1995).…”

Modulation of Allosteric Regulation by E38K and G101N Mutations in the Potato Tuber ADP-glucose Pyrophosphorylase

“…[11][12][13] In contrast to bacterial AGPase, a homotetrameric protein, the higher plant AGPase is composed of two small subunits (SSs) and two large subunits (LSs), which form a heterotetramer. [14][15][16] LS and SS share considerable sequence homology but have distinct roles in catalysis and allosteric regulation, as best exemplified by the potato tuber AGPase. Potato SS is capable of selfassembly to form an active enzyme.…”