Purification and Properties of Nonproteolytic Degraded ADPglucose Pyrophosphorylase from Maize Endosperm

Abstract

ADPglucose pyrophosphorylase from developing endosperm tissue of starchy maize (Zea mays) was purified 88-fold to a specific activity of 34 micromoles a-glucose-l-P produced per minute per milligram protein.Rabbit antiserum to purified spinach leaf ADPglucose pyrophosphorylase was able to inhibit pyrophosphorolysis activity of the purified enzyme by up to 90%. The final preparation yielded four major protein staining bands following sodium dodecyl sulfate polyacrylamide gel electrophoresis. When analyzed by Western blot hybridization only the fastest migrating, 54 kilodaltons, protein staining band cross-reacted with affinity purified rabbit antispinach leaf ADPglucose pyrophosphorylase immunoglobulin. The molecular mass of the native enzyme was estimated to be 230 kilodaltons. Thus, maize endosperm ADPglucose pyrophosphorylase appears to be comprised of four subunits. This is in contrast to the respective subunit and native molecular masses of 96 and 400 kilodaltons reported for a preparation of maize endosperm ADPglucose pyrophosphorylase (Fuchs RL and JO Smith 1979 Biochim Biophys Acta 556: 40-48). Proteolytic degradation of maize endosperm ADPglucose pyrophosphorylase appears to occur during incubation of crude extracts at 30°C or during the partial purification of the enzyme according to a previously reported procedure (DB Dickinson, J Preiss 1969 Arch Biochem Biophys 130: 119-128). The progressive appearance of a 53 kilodalton antigenic peptide suggested the loss of a 1 kilodalton proteolytic fragment from the 54 kilodalton subunit. The complete conservation of the 54 kilodalton subunit structure following extraction of the enzyme in the presence of phenylmethylsulfonyl fluoride and/or chymostatin was observed. The allosteric and catalytic properties of the partially purified proteolytic degraded versus nondegraded enzyme were compared. The major effect of proteolysis was to enhance enzyme activity in the absence of added activator while greatly decreasing its sensitivity to the allosteric effectors 3-P-glycerate and inorganic phosphate.Regulation of the activity of ADPglucose pyrophosphorylase (ATP:a-glucose-l-P adenyl transferase, EC 2.7.7.27) is believed to play a vital role in controlling the biosynthesis ofa-1 ,4-glucans in plants and bacteria (20,21). This enzyme catalyzes the reversible synthesis of ADPglucose and PP; from ATP and glucose-1-P and has had its kinetic and physical properties studied from a wide variety of sources (see Refs. 20-22, for reviews).

Multiple Forms of ADP-Glucose Pyrophosphorylase from Tomato Fruit

“…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). Although this phenomenon was observed in other purifications (Plaxton and Preiss, 1987;Iglesias et al, 1991), the increases by the heat treatment were usually not significant (below 1.5-fold). We consistently observed a 3-to 5-fold increase in enzyme activity by the heat treatment step.…”

“…Proteolytic degradation of the AGP small subunit was evident during the AGP purification from maize endosperm (Plaxton and Preiss, 1987;Preiss et al, 1989). 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).…”

“…Vol. 11 3 , 1997 chymostatin, the most effective protease inhibitors in tomato fruit crude extract judged by the Pep Tag protease assay, were also the only two effective protease inhibitors among 10 protease inhibitors tested in maize endosperm (Plaxton and Preiss, 1987).…”

“…The antibody against tomato fruit AGP was custom-made by the BAbCO-Berkeley Antibody Co. (Richmond, CA) with their standard protocol. Neutralization of AGI' activity was performed as previously described (Plaxton and Preiss, 1987).…”

“…The pyrophosphorylase of ADP-Glc was coupled with phosphoglucomutase and Glc-6-P dehydrogenase and assayed by measuring NADH production at 340 nm and 37°C (Plaxton and Preiss, 1987). A standard reaction mixture (1 mL) contained 80 pmol of Hepes-NaOH buffer (pH 7.4), 5 pmol of MgCI,, 10 pmol of 3-phosphoglycerate, 0.2 mg of BSA, 1 pmol of ADP-Glc, 1 pmol of sodium PPi, 0.6 pmol of NAD', 10 pmol of Glc-1,6-bisphosphate, and 2 units each of rabbit muscle phosphoglucomutase and Leuconostoc mesenteroides Glc-6-P dehydrogenase.…”