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Purification, oligomerization state and malate sensitivity of maize leaf phosphoenolpyruvate carboxylase.

ABSTRACT: A method was developed for the purification of phosphoenolpyruvate carboxylase from darkened maize leaves so that the enzyme retained its sensitivity to inhibition by malate. The procedure depended on the prevention of proteolysis by the inclusion of chymostatin in the buffers used during the purification. The purified enzyme was indistinguishable from that in crude extracts as judged by native polyacrylamide-gel electrophoresis. SDS/polyacrylamide-gel electrophoresis followed by immunoblotting, and Superose 6 gel filtration. Gel-filtration studies showed that the purified enzyme and the enzyme in extracts of darkened or illuminated leaves showed a concentration-dependent dissociation of tetrameric into dimeric forms. Purified phosphoenolpyruvate carboxylase and enzyme in crude extracts from darkened leaves were equally sensitive to inhibition by malate (Ki approx. 0.30 mM) under conditions where it existed in the tetrameric or dimeric forms, but the enzyme in crude extracts from illuminated leaves was less sensitive to malate inhibition (Ki approx. 0.95 mM) whether it was present as a tetramer or as a dimer. It is concluded that changes in the oligomerization state of phosphoenolpyruvate carboxylase are not directly involved in its regulation by light.

SUBMITTER: McNaughton GA

PROVIDER: S-EPMC1138832 | biostudies-other | 1989 Jul

REPOSITORIES: biostudies-other

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Project description:The photosynthetic phosphoenolpyruvate carboxylase isozyme from C4 plants (PEPC-C4) has a complex allosteric regulation, involving positive cooperativity in binding the substrate phosphoenolpyruvate as well as positive and negative allosteric effectors. Besides the proposed R- and T-states, previous kinetic results suggested functionally relevant different R-states of the maize enzyme (ZmPEPC-C4) elicited by PEP or its two kinds of activators, glucose 6-phosphate or glycine. To detect these different R-state conformations, we used as conformational probes the fluorescence of 8-anilino-1-naphthalene sulfonate (ANS), near-UV circular dichroism (CD) spectroscopy, and limited proteolysis by trypsin. Phosphoenolpyruvate and malate binding caused distinct concentration-dependent fluorescence changes of ZmPEPC-C4/ANS, suggesting that they elicited conformational states different from that of the free enzyme, while glucose 6-phosphate or glycine binding did not produce fluorescence changes. Differences were also observed in the near UV CD spectra of the enzyme, free or complexed with its substrate or allosteric effectors. Additionally, differences in the trypsin-digestion fragmentation patterns, as well as in the susceptibility of the free and complexed enzyme to digestion and digestion-provoked loss of activity, provided evidence of several ZmPEPC-C4 conformations in solution elicited by the substrate and the allosteric effectors. Using the already reported ZmPEPC-C4 crystal structures and bioinformatics methods, we predicted that the most probable trypsin-cleavage sites are located in superficial flexible regions, which seems relevant for the protein dynamics underlying the function and allosteric regulation of this enzyme. Together, our findings agree with previous kinetic results, shed light on this enzyme's complex allosteric regulation, and place ZmPEPC-C4 in the growing list of allosteric enzymes possessing an ensemble of closely related R-state conformations.

Project description:BackgroundAcetylation of promoter nucleosomes is tightly correlated and mechanistically linked to gene activity. However, transcription is not necessary for promoter acetylation. It seems, therefore, that external and endogenous stimuli control histone acetylation and by this contribute to gene regulation. Photosynthetic genes in plants are excellent models with which to study the connection between stimuli and chromatin modifications because these genes are strongly expressed and regulated by multiple stimuli that are easily manipulated. We have previously shown that acetylation of specific histone lysine residues on the photosynthetic phosphoenolpyruvate carboxylase (Pepc) promoter in maize is controlled by light and is independent of other stimuli or gene activity. Acetylation of upstream promoter regions responds to a set of other stimuli which include the nutrient availability of the plant. Here, we have extended these studies by analysing histone acetylation during the diurnal and circadian rhythm of the plant.ResultsWe show that histone acetylation of individual lysine residues is removed from the core promoter before the end of the illumination period which is an indication that light is not the only factor influencing core promoter acetylation. Deacetylation is accompanied by a decrease in gene activity. Pharmacological inhibition of histone deacetylation is not sufficient to prevent transcriptional repression, indicating that deacetylation is not controlling diurnal gene regulation. Variation of the Pepc promoter activity during the day is controlled by the circadian oscillator as it is maintained under constant illumination for at least 3 days. During this period, light-induced changes in histone acetylation are completely removed from the core promoter, although the light stimulus is continuously applied. However, acetylation of most sites on upstream promoter elements follows the circadian rhythm.ConclusionOur results suggest a central role of upstream promoter acetylation in the quantitative regulation of gene expression in this model gene. Induced core promoter acetylation is dispensable for the highest gene expression in the diurnal and circadian rhythm.

Project description:Phosphoenolpyruvate carboxylase (PEPC) is an important enzyme in plants, which regulates carbon flow through the TCA cycle and controls protein and oil biosynthesis. Although it is important, there is little research on PEPC in cotton, the most important fiber crop in the world. In this study, a total of 125 PEPCs were identified in 15 Gossypium genomes. All PEPC genes in cotton are divided into six groups and each group generally contains one PEPC member in each diploid cotton and two in each tetraploid cotton. This suggests that PEPC genes already existed in cotton before their divergence. There are additional PEPC sub-groups in other plant species, suggesting the different evolution and natural selection during different plant evolution. PEPC genes were independently evolved in each cotton sub-genome. During cotton domestication and evolution, certain PEPC genes were lost and new ones were born to face the new environmental changes and human being needs. The comprehensive analysis of collinearity events and selection pressure shows that genome-wide duplication and fragment duplication are the main methods for the expansion of the PEPC family, and they continue to undergo purification selection during the evolutionary process. PEPC genes were widely expressed with temporal and spatial patterns. The expression patterns of PEPC genes were similar in G. hirsutum and G. barbadense with a slight difference. PEPC2A and 2D were highly expressed in cotton reproductive tissues, including ovule and fiber at all tested developmental stages in both cultivated cottons. However, PEPC1A and 1D were dominantly expressed in vegetative tissues. Abiotic stress also induced the aberrant expression of PEPC genes, in which PEPC1 was induced by both chilling and salinity stresses while PEPC5 was induced by chilling and drought stresses. Each pair (A and D) of PEPC genes showed the similar response to cotton development and different abiotic stress, suggesting the similar function of these PEPCs no matter their origination from A or D sub-genome. However, some divergence was also observed among their origination, such as PEPC5D was induced but PEPC5A was inhibited in G. barbadense during drought treatment, suggesting that a different organized PEPC gene may evolve different functions during cotton evolution. During cotton polyploidization, the homologues genes may refunction and play different roles in different situations.

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Purification, oligomerization state and malate sensitivity of maize leaf phosphoenolpyruvate carboxylase.

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