The bacterial-type phosphoenolpyruvate carboxylase isozyme from developing castor oil seeds is subject to in vivo regulatory phosphorylation at serine-451

Phosphoenolpyruvate carboxylase (PEPC) is a tightly controlled anaplerotic enzyme situated at a pivotal branch point of plant carbohydrate-metabolism. In developing castor oil seeds (COS) a novel allosterically-densensitized 910-kDa Class-2 PEPC hetero-octameric complex arises from a tight interaction between 107-kDa plant-type PEPC and 118-kDa bacterial-type PEPC (BTPC) subunits. Mass spectrometry and immunoblotting with anti-phosphoSer451 specific antibodies established that COS BTPC is in vivo phosphorylated at Ser451, a highly conserved target residue that occurs within an intrinsically disordered region. This phosphorylation was enhanced during COS development or in response to depodding. Kinetic characterization of a phosphomimetic (S451D) mutant indicated that Ser451 phosphorylation inhibits the catalytic activity of BTPC subunits within the Class-2 PEPC complex.     

Coimmunopurification of phosphorylated bacterial- and plant-type phosphoenolpyruvate carboxylases with the plastidial pyruvate dehydrogenase complex from developing castor oil seeds

The phosphoenolpyruvate carboxylase (PEPC) interactome of developing castor oil seed (COS; Ricinus communis) endosperm was assessed using coimmunopurification (co-IP) followed by proteomic analysis. Earlier studies suggested that immunologically unrelated 107-kD plant-type PEPCs (p107/PTPC) and 118-kD bacterial-type PEPCs (p118/BTPC) are subunits of an unusual 910-kD hetero-octameric class 2 PEPC complex of developing COS. The current results confirm that a tight physical interaction occurs between p118 and p107 because p118 quantitatively coimmunopurified with p107 following elution of COS extracts through an anti-p107-IgG immunoaffinity column. No PEPC activity or immunoreactive PEPC polypeptides were detected in the corresponding flow-through fractions. Although BTPCs lack the N-terminal phosphorylation motif characteristic of PTPCs, Pro-Q Diamond phosphoprotein staining, immunoblotting with phospho-serine (Ser)/threonine Akt substrate IgG, and phosphate-affinity PAGE established that coimmunopurified p118 was multiphosphorylated at unique Ser and/or threonine residues. Tandem mass spectrometric analysis of an endoproteinase Lys-C p118 peptide digest demonstrated that Ser-425 is subject to in vivo proline-directed phosphorylation. The co-IP of p118 with p107 did not appear to be influenced by their phosphorylation status. Because p118 phosphorylation was unchanged 48 h following elimination of photosynthate supply due to COS depodding, the signaling mechanisms responsible for photosynthate-dependent p107 phosphorylation differ from those controlling p118's in vivo phosphorylation. A 110-kD PTPC coimmunopurified with p118 and p107 when depodded COS was used. The plastidial pyruvate dehydrogenase complex (PDC(pl)) was identified as a novel PEPC interactor. Thus, a putative metabolon involving PEPC and PDC(pl) could function to channel carbon from phosphoenolpyruvate to acetyl-coenzyme A and/or to recycle CO(2) from PDC(pl) to PEPC.     

Phosphorylation of bacterial-type phosphoenolpyruvate carboxylase at Ser425 provides a further tier of enzyme control in developing castor oil seeds

PEPC [PEP (phosphoenolpyruvate) carboxylase] is a tightly controlled anaplerotic enzyme situated at a pivotal branch point of plant carbohydrate metabolism. Two distinct oligomeric PEPC classes were discovered in developing COS (castor oil seeds). Class-1 PEPC is a typical homotetramer of 107?kDa PTPC (plant-type PEPC) subunits, whereas the novel 910-kDa Class-2 PEPC hetero-octamer arises from a tight interaction between Class-1 PEPC and 118?kDa BTPC (bacterial-type PEPC) subunits. Mass spectrometric analysis of immunopurified COS BTPC indicated that it is subject to in vivo proline-directed phosphorylation at Ser425. We show that immunoblots probed with phosphorylation site-specific antibodies demonstrated that Ser425 phosphorylation is promoted during COS development, becoming maximal at stage IX (maturation phase) or in response to depodding. Kinetic analyses of a recombinant, chimaeric Class-2 PEPC containing phosphomimetic BTPC mutant subunits (S425D) indicated that Ser425 phosphorylation results in significant BTPC inhibition by: (i) increasing its Km(PEP) 3-fold, (ii) reducing its I50 (L-malate and L-aspartate) values by 4.5- and 2.5-fold respectively, while (iii) decreasing its activity within the physiological pH range. The developmental pattern and kinetic influence of Ser425 BTPC phosphorylation is very distinct from the in vivo phosphorylation/activation of COS Class-1 PEPC's PTPC subunits at Ser11. Collectively, the results establish that BTPC's phospho-Ser425 content depends upon COS developmental and physiological status and that Ser425 phosphorylation attenuates the catalytic activity of BTPC subunits within a Class-2 PEPC complex. To the best of our knowledge, this study provides the first evidence for protein phosphorylation as a mechanism for the in vivo control of vascular plant BTPC activity.     

The remarkable diversity of plant PEPC (phosphoenolpyruvate carboxylase): recent insights into the physiological functions and post-translational controls of non-photosynthetic PEPCs

PEPC [PEP (phosphoenolpyruvate) carboxylase] is a tightly controlled enzyme located at the core of plant C-metabolism that catalyses the irreversible β-carboxylation of PEP to form oxaloacetate and Pi. The critical role of PEPC in assimilating atmospheric CO(2) during C(4) and Crassulacean acid metabolism photosynthesis has been studied extensively. PEPC also fulfils a broad spectrum of non-photosynthetic functions, particularly the anaplerotic replenishment of tricarboxylic acid cycle intermediates consumed during biosynthesis and nitrogen assimilation. An impressive array of strategies has evolved to co-ordinate in vivo PEPC activity with cellular demands for C(4)-C(6) carboxylic acids. To achieve its diverse roles and complex regulation, PEPC belongs to a small multigene family encoding several closely related PTPCs (plant-type PEPCs), along with a distantly related BTPC (bacterial-type PEPC). PTPC genes encode ~110-kDa polypeptides containing conserved serine-phosphorylation and lysine-mono-ubiquitination sites, and typically exist as homotetrameric Class-1 PEPCs. In contrast, BTPC genes encode larger ~117-kDa polypeptides owing to a unique intrinsically disordered domain that mediates BTPC's tight interaction with co-expressed PTPC subunits. This association results in the formation of unusual ~900-kDa Class-2 PEPC hetero-octameric complexes that are desensitized to allosteric effectors. BTPC is a catalytic and regulatory subunit of Class-2 PEPC that is subject to multi-site regulatory phosphorylation in vivo. The interaction between divergent PEPC polypeptides within Class-2 PEPCs adds another layer of complexity to the evolution, physiological functions and metabolic control of this essential CO(2)-fixing plant enzyme. The present review summarizes exciting developments concerning the functions, post-translational controls and subcellular location of plant PTPC and BTPC isoenzymes.     

In vivo regulatory phosphorylation of novel phosphoenolpyruvate carboxylase isoforms in endosperm of developing castor oil seeds

Our previous research characterized two phosphoenolpyruvate (PEP) carboxylase (PEPC) isoforms (PEPC1 and PEPC2) from developing castor oil seeds (COS). The association of a shared 107-kD subunit (p107) with an immunologically unrelated bacterial PEPC-type 64-kD polypeptide (p64) leads to marked physical and kinetic differences between the PEPC1 p107 homotetramer and PEPC2 p107/p64 heterooctamer. Here, we describe the production of antiphosphorylation site-specific antibodies to the conserved p107 N-terminal serine-6 phosphorylation site. Immunoblotting established that the serine-6 of p107 is phosphorylated in COS PEPC1 and PEPC2. This phosphorylation was reversed in vitro following incubation of clarified COS extracts or purified PEPC1 or PEPC2 with mammalian protein phosphatase type 2A and is not involved in a potential PEPC1 and PEPC2 interconversion. Similar to other plant PEPCs examined to date, p107 phosphorylation increased PEPC1 activity at pH 7.3 by decreasing its K(m)(PEP) and sensitivity to L-malate inhibition, while enhancing glucose-6-P activation. By contrast, p107 phosphorylation increased PEPC2's K(m)(PEP) and sensitivity to malate, glutamic acid, and aspartic acid inhibition. Phosphorylation of p107 was promoted during COS development (coincident with a >5-fold increase in the I(50) [malate] value for total PEPC activity in desalted extracts) but disappeared during COS desiccation. The p107 of stage VII COS became fully dephosphorylated in planta 48 h following excision of COS pods or following 72 h of dark treatment of intact plants. The in vivo phosphorylation status of p107 appears to be modulated by photosynthate recently translocated from source leaves into developing COS.     

Bacterial- and plant-type phosphoenolpyruvate carboxylase isozymes from developing castor oil seeds interact in vivo and associate with the surface of mitochondria

Phosphoenolpyruvate carboxylase (PEPC) from developing castor oil seeds (COS) exists as two distinct oligomeric isoforms. The typical class-1 PEPC homotetramer consists of 107-kDa plant-type PEPC (PTPC) subunits, whereas the allosterically desensitized 910-kDa class-2 PEPC hetero-octamer arises from the association of class-1 PEPC with 118-kDa bacterial-type PEPC (BTPC) subunits. The in vivo interaction and subcellular location of COS BTPC and PTPC were assessed by imaging fluorescent protein (FP)-tagged PEPCs in tobacco suspension-cultured cells. The BTPC-FP mainly localized to cytoplasmic punctate/globular structures, identified as mitochondria by co-immunostaining of endogenous cytochrome oxidase. Inhibition of respiration with KCN resulted in proportional decreases and increases in mitochondrial versus cytosolic BTPC-FP, respectively. The FP-PTPC and NLS-FP-PTPC (containing an appended nuclear localization signal, NLS) localized to the cytosol and nucleus, respectively, but both co-localized with mitochondrial-associated BTPC when co-expressed with BTPC-FP. Transmission electron microscopy of immunogold-labeled developing COS revealed that BTPC and PTPC are localized at the mitochondrial (outer) envelope, as well as the cytosol. Moreover, thermolysin-sensitive BTPC and PTPC polypeptides were detected on immunoblots of purified COS mitochondria. Overall, our results demonstrate that: (i) COS BTPC and PTPC interact in vivo as a class-2 PEPC complex that associates with the surface of mitochondria, (ii) BTPC's unique and divergent intrinsically disordered region mediates its interaction with PTPC, whereas (iii) the PTPC-containing class-1 PEPC is entirely cytosolic. We hypothesize that mitochondrial-associated class-2 PEPC facilitates rapid refixation of respiratory CO(2) while sustaining a large anaplerotic flux to replenish tricarboxylic acid cycle C-skeletons withdrawn for biosynthesis.     

Structural and kinetic properties of high and low molecular mass phosphoenolpyruvate carboxylase isoforms from the endosperm of developing castor oilseeds

Phosphoenolpyruvate carboxylase (PEPC) is believed to play an important role in producing malate as a substrate for fatty acid synthesis by leucoplasts of the developing castor oilseed (COS) endosperm. Two kinetically distinct isoforms of COS PEPC were resolved by gel filtration chromatography and purified. PEPC1 is a typical 410-kDa homotetramer composed of 107-kDa subunits (p107). In contrast, PEPC2 exists as an unusual 681-kDa hetero-octamer composed of the same p107 found in PEPC1 and an associated 64-kDa polypeptide (p64) that is structurally and immunologically unrelated to p107. Relative to PEPC1, PEPC2 demonstrated significantly enhanced thermal stability and a much lower sensitivity to allosteric activators (Glc-6-P, Glc-1-P, Fru-6-P, glycerol-3-P) and inhibitors (Asp, Glu, malate) and pH changes within the physiological range. Nondenaturing PAGE of clarified extracts followed by in-gel PEPC activity staining indicated that the ratio of PEPC1:PEPC2 increases during COS development such that only PEPC1 is detected in mature COS. Dissimilar developmental profiles and kinetic properties support the hypotheses that (i) PEPC1 functions to replenish dicarboxylic acids consumed through transamination reactions required for storage protein synthesis, whereas (ii) PEPC2 facilitates PEP flux to malate in support of fatty acid synthesis. Interestingly, the respective physical and kinetic properties of COS PEPC1 and PEPC2 are remarkably comparable with those of the homotetrameric low M(r) Class 1 and heteromeric high M(r) Class 2 PEPC isoforms of unicellular green algae.     

Two unrelated phosphoenolpyruvate carboxylase polypeptides physically interact in the high molecular mass isoforms of this enzyme in the unicellular green alga Selenastrum minutum

In the chlorophyte Selenastrum minutum, phosphoenolpyruvate carboxylase (PEPC) exists as two kinetically distinct classes of isoforms sharing the same 102-kDa catalytic subunit (p102). Class 1 PEPC is homotetrameric, whereas Class 2 PEPCs consist of three large protein complexes. The different Class 2 PEPCs contain p102 and 130-, 73-, and 65-kDa polypeptides in different stoichiometric combinations. Immunoblot, immunoprecipitation, and chemical cross-linking studies indicated that p102 physically interacts with the 130-kDa polypeptide (p130) in Class 2 PEPCs. Immunological data and mass spectrometric and sequence analyses revealed that p102 and p130 are not closely related even if a p130 tryptic peptide had significant similarity to a conserved PEPC C-terminal domain from several sources. Evidence supporting the hypothesis that p130 has PEPC activity includes the following. (i) Specific activity expressed relative to the amount of p102 was lower in Class 1 than in Class 2 PEPCs; (ii) reductive pyridoxylation of both p102 and p130 was inhibited by magnesium-phosphoenolpyruvate; and (iii) biphasic phosphoenolpyruvate binding kinetics were observed with Class 2 PEPCs. These data support the view that unicellular green algae uniquely express, regulate, and assemble divergent PEPC polypeptides. This probably serves an adaptive purpose by poising these organisms for survival in different environments varying in nutrient content.     

In vitro proteolysis of phosphoenolpyruvate carboxylase from developing castor oil seeds by an endogenous thiol endopeptidase

Two novel phosphoenolpyruvate carboxylase (PEPC) isoforms have been biochemically characterized from endosperm of developing castor oil seeds (COS). The association of a 107 kDa PEPC subunit (p107) with an immunologically unrelated bacterial PEPC-type 64 kDa polypeptide leads to marked physical and kinetic differences between the PEPC1 p107 homotetramer and PEPC2 p107/p64 heterooctamer. COS p107 is quite susceptible to limited proteolysis during PEPC purification. An endogenous asparaginyl endopeptidase appears to catalyze the in vitro cleavage of an approximately 120 amino acid polypeptide from the N-terminal end of p107, producing a truncated 98 kDa polypeptide (p98). Immunoblotting was used to estimate proteolytic activity by following the disappearance of p107 and concomitant appearance of p98 during incubation of clarified COS extracts at 4 degrees C. The in vitro proteolysis of p107 to p98 only occurred in the combined presence of 2 mM dithiothreitol and high salt concentrations (particularly SO(4) (2-) and PO(4) (2-) salts). Although p107-degrading activity was present throughout COS development, it was most pronounced in endosperm extracts from older beans. Several protease inhibitors, including two commercially available protease inhibitor cocktails, were tested for their ability to prevent p107 proteolysis. All of the inhibitors were ineffective except for 2,2'-dipyridyl disulfide (DPDS), a relatively inexpensive and underutilized active site inhibitor of plant thiol proteases. Asparaginyl endopeptidase activity of COS extracts was unaffected by 20% (NH(4))(2)SO(4) when determined in the presence or absence of 2 mM dithiothreitol using a spectrophotometric assay based upon the hydrolysis of benzoyl-L-Asn-p-nitroanilide. Thus, we propose that the combined presence of 2 mM dithiothreitol and 20% (NH(4))(2)SO(4) promotes a p107 conformational change that exposes the N-terminal region asparaginyl residue where p107 hydrolysis is believed to occur.     

Phospho<Emphasis Type="Italic">enol</Emphasis>pyruvate carboxylase protein kinase from developing castor oil seeds: partial purification,characterization, and reversible control by photosynthate supply

Phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) protein kinase (PPCK) was purified ∼1,500-fold from developing castor oil seeds (COS). Gel filtration and immunoblotting with anti-(rice PPCK2)-immune serum indicated that this Ca²⁺-insensitive PPCK exists as a 31-kDa monomer. COS PPCK-mediated rephosphorylation of the 107-kDa subunit (p107) of COS PEPC1 (K _m = 2.2 μM) activated PEPC1 by ∼80% when assayed under suboptimal conditions (pH 7.3, 0.2 mM PEP, and 0.125 mM malate). COS PPCK displayed remarkable selectivity for phosphorylating COS PEPC1 (relative to tobacco, sorghum, or maize PEPCs), exhibited a broad pH-activity optima of ∼pH 8.5, and at pH 7.3 was activated 40–65% by 1 mM PEP, or 10 mM Gln or Asn, but inhibited 65% by 10 mM L-malate. The possible control of COS PPCK by disulfide-dithiol interconversion was suggested by its rapid inactivation and subsequent reactivation when incubated with oxidized glutathione and then dithiothreitol. In vitro PPCK activity correlated with in vivo p107 phosphorylation status, with both peaking in mid-cotyledon to full-cotyledon developing COS. Notably, PPCK activity and p107 phosphorylation of developing COS were eliminated following pod excision or prolonged darkness of intact plants. Both effects were fully reversed 12 h following reillumination of darkened plants. These results implicate a direct relationship between the up-regulation of COS PPCK and p107 phosphorylation during the recommencement of photosynthate delivery from illuminated leaves to the non-photosynthetic COS. Overall, the results support the hypothesis that PEPC and PPCK participate in the control of photosynthate partitioning into C-skeletons needed as precursors for key biosynthetic pathways of developing COS. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Characterization of bacterial-type phospho<Emphasis Type="Italic">enol</Emphasis>pyruvate carboxylase expressed in male gametophyte of higher plants

Background  

Phosphoenolpyruvate carboxylase (PEPC) is a critical enzyme catalyzing the β-carboxylation of phosphoenolpyruvate (PEP) to oxaloacetate, a tricarboxylic acid (TCA) cycle intermediate. PEPC typically exists as a Class-1 PEPC homotetramer composed of plant-type PEPC (PTPC) polypeptides, and two of the subunits were reported to be monoubiquitinated in germinating castor oil seeds. By the large-scale purification of ubiquitin (Ub)-related proteins from lily anther, two types of PEPCs, bacterial-type PEPC (BTPC) and plant-type PEPC (PTPC), were identified in our study as candidate Ub-related proteins. Until now, there has been no information about the properties of the PEPCs expressed in male reproductive tissues of higher plants.     

In vitro phosphorylation of phosphoenolpyruvate carboxylase from the green alga Selenastrum minutum

Previously, we described two distinct classes of phosphoenolpyruvate carboxylase (PEPC) isoforms in the green alga Selenastrum minutum. Class 1 PEPC (PEPC1) is a homotetramer composed of 102 kDa subunits (p102), whereas Class 2 PEPCs exist as three large protein complexes (PEPC2-PEPC4) containing varying proportions of structurally dissimilar p102 and 130 kDa (p130) PEPC catalytic subunits. In the current study, a p102 calcium-independent protein kinase was shown to co-purify with PEPC1, but not PEPC2. However, the p130 subunit of PEPC2 was phosphorylated in vitro during its incubation in the presence of [gamma-(32)P]ATP and a clarified algal extract. Treatment of purified PEPC2 with protein phosphatase 2A(2) increased its apparent M(r) as judged by Superose 6 gel filtration chromatography. The presence of the protein phosphatase inhibitors NaF and microcystin-LR throughout PEPC purification significantly influenced the activity and structural organization of Class 2, but not Class 1, PEPC isoforms. The results are consistent with the notion that under the culture conditions employed: (i) Class 1 and Class 2 PEPC isoforms exist in vivo mainly in their dephosphorylated and phosphorylated forms, respectively, and (ii) phosphorylation of Class 2 PEPCs leads to a significant reduction in their activity and native M(r). We propose that protein kinase-mediated phosphorylation is involved in the control and structural organization of green algal PEPC.     

Purification and characterization of phosphoenolpyruvate carboxylase from Brassica napus (rapeseed) suspension cell cultures: implications for phosphoenolpyruvate carboxylase regulation during phosphate starvation, and the integration of glycolysis with nitrogen assimilation.

Phosphoenolpyruvate carboxylase (PEPC) specific activity increased by 250% following 8 to 10 days of Pi starvation of Brassica napus suspension cells. Densitometric scanning of PEPC immunoblots revealed a close correlation between PEPC activity and the amount of the antigenic 104-kDa PEPC subunit. To further assess the influence of Pi deprivation on PEPC, the enzyme was purified from Pi-sufficient (+Pi) and Pi-starved (-Pi) cells to electrophoretic homogeneity and final specific activities of 37-40 micromol phosphoenolpyruvate utilized per min per mg protein. Gel filtration, SDS/PAGE, and CNBr peptide mapping indicated that the +Pi and -Pi PEPCs are both homotetramers composed of an identical 104-kDa subunit. Respective pH-activity profiles, phosphoenolpyruvate saturation kinetics, and sensitivity to L-malate inhibition were also indistinguishable. Kinetic studies and phosphatase treatments revealed that PEPC of the +Pi and -Pi cells exists mainly in its dephosphorylated (L-malate sensitive) form. Thus, up-regulation of PEPC activity in -Pi cells appears to be solely due to the accumulation of the same PEPC isoform being expressed in +Pi cells. PEPC activity was modulated by several metabolites involved in carbon and nitrogen metabolism. At pH 7.3, marked activation by glucose 6-phosphate and inhibition by L-malate, L-aspartate, L-glutamate, DL-isocitrate, rutin and quercetin was observed. The following paper provides a model for the coordinate regulation of B. napus PEPC and cytosolic pyruvate kinase by allosteric effectors. L-Aspartate and L-glutamate appear to play a crucial role in the control of the phosphoenolpyruvate branchpoint in B. napus, particularly with respect to the integration of carbohydrate partitioning with the generation of carbon skeletons required during nitrogen assimilation.     

Comparative studies of phosphoenolpyruvate carboxylase from c(3) and c(4) plants

Phosphoenolpyruvate carboxylase (PEPC) from several C₃ plants was compared to maize PEPC by immunoblotting using an antibody against maize PEPC and by peptide mapping. In C₃ gramineous plants, PEPCs of slightly different monomeric sizes were detected as two bands for wheat and barley leaves, as three bands for etiolated maize leaves and as four bands for rice leaves by SDS-polyacrylamide gel electrophoresis and immunoblotting, whereas only one PEPC band was detected for maize leaves, a C₄ plant, or tobacco leaves, a dicotyledonous C₃ plant. The peptide fragment patterns of the lower molecular weight PEPC (major band in immunoblotting) in wheat leaves was similar to that of maize PEPC in peptide mapping by protein staining or by immunological detection, but the upper one (minor band) had a different pattern from the lower one in peptide mapping by immunological detection and few peptide fragments from this were recognized by the anti-(maize) PEPC antibody. These results suggest that there are multiple forms of PEPC subunits in the gramineous plants tested, and the major PEPC has a primary structure similar to that of maize PEPC. To obtain information about the expression of PEPCs in C₃ plants, changes in the amount of PEPC protein were investigated during the greening of rice and wheat seedlings. Judging from the regulation by light, there were two types of PEPCs in greening rice seedlings, one induced by light and the other reduced by it. Greening wheat seedlings also show a PEPC band induced by light. These findings indicate that some PEPCs in C₃ gramineous plants not only have structures similar to that of maize PEPC, but also are regulated by light in a similar manner.     

In Vivo and in Vitro Phosphorylation of the Phosphoenolpyruvate Carboxylase from Wheat Seeds during Germination

Phosphoenolpyruvate carboxylase (PEPC) activity was detected in the aleurone endosperm of wheat (Triticum aestivum cv Chinese Spring) seeds, and specific anti-Sorghum C4 PEPC polyclonal anti-bodies cross-reacted with 103- and 100-kD polypeptides present in dry seeds and seeds that had imbibed; in addition, a new, 108-kD polypeptide was detected 6 h after imbibition. The use of specific anti-phosphorylation-site immunoglobulin G (APS-IgG) identified the presence of a phosphorylation motif equivalent to that found in other plant PEPCs studied so far. The binding of this APS-IgG to the target protein promoted changes in the properties of seed PEPC similar to those produced by phosphorylation, as previously shown for the recombinant Sorghum leaf C4 PEPC. In desalted seed extracts, an endogenous PEPC kinase activity catalyzed a bona fide phosphorylation of the target protein, as deduced from the immunoinhibition of the in vitro phosphorylation reaction by the APS- IgG. In addition, the major, 103-kD PEPC polypeptide was also shown to be radiolabeled in situ 48 h after imbibition in [32P]orthophosphate. The ratio between optimal (pH 8) and suboptimal (pH 7.3 or 7.1) PEPC activity decreased during germination, thereby suggesting a change in catalytic rate related to an in vivo phosphorylation process. These collective data document that the components needed for the regulatory phosphorylation of PEPC are present and functional during germination of wheat seeds.