Stephen M. G. Duff

In Vivo Regulation of Wheat-Leaf Phosphoenolpyruvate Carboxylase by Reversible Phosphorylation

Regulation of C3 phosphoenolpyruvate carboxylase (PEPC) and its protein-serine/threonine kinase (PEPC-PK) was studied in wheat (Triticum aestivum) leaves that were excised from low-N-grown seedlings and subsequently illuminated and/or supplied with 40 mM KNO3. The apparent phosphorylation status of PEPC was assessed by its sensitivity to L-malate inhibition at suboptimal assay conditions, and the activity state of PEPC-PK was determined by the in vitro 32P labeling of purified maize dephospho-PEPC by [[gamma]-32P]ATP/Mg. Illumination ([plus or minus]NO3-) for 1 h led to about a 4.5-fold increase in the 50% inhibition constant for L-malate, which was reversed by placing the illuminated detached leaves in darkness (minus NO3-). A 1 -h exposure of excised leaves to light, KNO3, or both resulted in relative PEPC-PK activities of 205, 119, and 659%, respectively, of the dark/0 mM KNO3 control tissue. In contrast, almost no activity was observed when a recombinant sorghum phosphorylation-site mutant (S8D) form of PEPC was used as protein substrate in PEPC-PK assays of the light plus KNO3 leaf extracts. In vivo labeling of wheat-leaf PEPC by feeding 32P-labeled orthophosphate showed that PEPC from light plus KNO3 tissue was substantially more phosphorylated than the enzyme in the dark minus-nitrate immunoprecipitates. Immunoblot analysis indicated that no changes in relative PEPC-protein amount occurred within 1 h for any of the treatments. Thus, C3 PEPC activity in these detached wheat leaves appears to be regulated by phosphorylation of a serine residue near the proteins N terminus by a Ca2+ -independent protein kinase in response to a complex interaction in vivo between light and N.

Purification and Characterization of a Potato Tuber Acid Phosphatase Having Significant Phosphotyrosine Phosphatase Activity.

The major acid phosphatase (APase) from potato (Solanum tuberosom L. cv Chiefton) tubers has been purified 2289-fold to near homogeneity and a final O-phospho-L-tyrosine (P-Tyr) hydrolyzing specific activity of 1917 [mu]mol Pi produced min-1 mg-1 of protein. Nondenaturing polyacrylamide gel electrophoresis of the final preparation resolved a single protein-staining band that co-migrated with APase activity. Following sodium dodecyl sulfate polyacrylamide gel electrophoresis, glycosylated polypeptides of 57 and 55 kD were observed. The two polypeptides are immunologically closely related, since both proteins cross-reacted on immunoblots probed with rabbit anti-(Brassica nigra APase) immunoglobulin G. Immunoblotting studies revealed that the 55-kD subunit did not arise via proteolytic cleavage of the 57-kD subunit after tissue extraction. The native molecular mass was approximately 100 kD, suggesting that the holoenzyme could exist as either a homodimer or a heterodimer. The enzyme displayed a pH optimum of 5.8, was activated 40% by 4 mM Mg2+, and was potently inhibited by molybdate, vanadate, and ZnCl2. The final preparation displayed the highest activity and specificity constant with P-Tyr, but also dephosphorylated other phosphomonoesters including p-nitrophenylphosphate, O-phospho-L-serine, phosphoenolpyruvate, PPi, and ATP. Antibodies to P-Tyr were used to demonstrate that several endogenous phosphotyrosylated tuber polypeptides could serve as in vitro substrates for the purified APase. Although the precise physiological significance of the potato APases substantial in vitro activity with P-Tyr remains obscure, the possibility that this APase may function to dephosphorylate certain protein-located P-Tyr residues in vivo is suggested.

Purification, characterization, and subcellular localization of an acid phosphatase from black mustard cell-suspension cultures: Comparison with phosphoenolpyruvate phosphatase☆

An acid phosphatase from Brassica nigra (black mustard) leaf petiole cell-suspension cultures has been purified 1633-fold to a final specific activity of 1225 (mumols orthophosphate produced/min)/mg protein and near homogeneity. The native protein was a glycosylated monomer having a molecular mass of 60 kDa and a pI of 4.5. The enzyme displayed a broad pH optimum of about pH 5.6 and was heat stable. The final preparation hydrolyzed a wide variety of phosphate esters. The highest specificity constants were obtained with 3-phosphoglycerate, 2,3-diphosphoglycerate, PPi, and phosphoenolpyruvate (PEP). The enzyme was activated 1.4-fold by 4 mM Mg2+ or Mn2+, but was strongly inhibited by Mo, Pi, F, and several phosphorylated compounds. Subcellular localization experiments revealed that this nonspecific acid phosphatase is probably a secreted enzyme, localized in the cell wall. By contrast, B. nigra PEP phosphatase appeared to be localized in the cell vacuole. Peptide mapping via CNBr fragmentation was employed to investigate the structural relatedness of the two phosphatases. Their respective CNBr cleavage patterns were dissimilar, suggesting that B. nigra acid and PEP phosphatases are distinct polypeptides. Putative metabolic functions of these two phosphatases are discussed in relation to the biochemical adaptations of B. nigra cell-suspension cultures to nutritional phosphate deprivation.

In-situ evidence for the involvement of calcium and bundle-sheath-derived photosynthetic metabolites in the C4 phosphoenolpyruvate-carboxylase kinase signal-transduction chain

Regulation of the light activation of C4 phosphoenolpyruvate-carboxylase (PEPC) protein kinase (PEPC-PK) and the ensuing phosphorylation of its cytosolic target protein were studied in intact mesophyll cells (MC) and protoplasts (MP) isolated from dark-adapted leaves of Digitaria sanguinalis [L.] Scop, (hairy crabgrass). The apparent in-situ phosphorylation state of PEPC (EC 4.1.1.31) was assessed by the sensitivity of its activity in desalted MC- and MP-extracts to l-malate under suboptimal assay conditions, while the activity-state of PEPC-PK was determined by in-vitro 32P-labeling of purified maize or recombinant sorghum PEPC by these extracts. In-situ pretreatment of intact MC at pH 8.0 by illumination and calcium addition led to significant decreases in PEPC malate sensitivity and increases in PEPC-kinase activity that were negated by the addition of EGTA to the external cell medium. Similarly, in-situ pretreatment of MP with light plus NH4Cl at pH 7.6 led to significant decreases in malate sensitivity which did not occur when a Ca2+ ionophore and EGTA were included in the suspension medium. In contrast, neither EGTA nor exogenous Ca2+ had a major direct effect on the in-vitro activity of PEPC-PK extracted from Digitaria MC and MP. Preincubation of intact MC with 5 mM 3-phosphoglycerate or pyruvate at pH 8.0 in the dark led to significant decreases in PEPC malate sensitivity and increases in PEPC-PK activity which were not observed with various other exogenous metabolites. These collective in-situ experiments with isolated C4 MC and MP (i) support our earlier hypothesis that alkalization of cytosolic pH is involved in the PEPC-PK signal-transduction cascade (see J.-N. Pierre et al., Eur J Biochem, 1992,210: 531–537), (ii) suggest that intracellular calcium is involved in the PEPC-kinase signal-transduction chain, but at a step upstream of PEPC-PK per se, and (iii) provide direct evidence that the bundle-sheath-derived, C4-pathway intermediates 3-PGA and/or pyruvate also play a role in this signal-transduction cascade which ultimately effects the up-regulation of PEPC in the C4 mesophyll cytosol.