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1.
Arch Biochem Biophys ; 375(1): 165-70, 2000 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-10683263

RESUMO

In C(4) plants such as maize, pyruvate,orthophosphate dikinase (PPDK) catalyzes the regeneration of the initial carboxylation substrate during C(4) photosynthesis. The primary catalytic residue, His-458 (maize C(4) PPDK), is involved in the ultimate transfer of the beta-phosphate from ATP to pyruvate. C(4) PPDK activity undergoes light-dark regulation in vivo by reversible phosphorylation of a nearby active-site residue (Thr-456) by a single bifunctional regulatory protein (RP). Using site-directed mutagenesis of maize recombinant C(4) dikinase, we made substitutions at the catalytic His residue (H458N) and at this regulatory target Thr (T456E, T456Y, T456F). Each of these affinity-purified mutant enzymes was assayed for changes in dikinase activity. As expected, substituting His-458 with Asn results in a catalytically incompetent enzyme. Substitutions of the Thr-456 residue with Tyr and Phe reduced activity by about 94 and 99%, respectively. Insertion of Glu at this position completely abolished activity, presumably by the introduction of negative charge proximal to the catalytic His. Furthermore, neither the T456Y nor inactive H458N mutant enzyme was phosphorylated in vitro by RP. The inability of the former to serve as a phosphorylation substrate indicates that RP is functionally a member of the Ser/Thr family of protein kinases rather than a "dual-specificity" Ser-Thr/Tyr kinase, since our previous work showed that RP effectively phosphorylated Ser inserted at position 456. The inability of RP to phosphorylate its native target Thr residue when Asn is substituted for His-458 documents that RP requires the His-P catalytic intermediate form of PPDK as its protein substrate. For these latter studies, synthetic phosphopeptide-directed antibodies specific for the Thr(456)-P form of maize C(4) PPDK were developed and characterized.


Assuntos
Domínio Catalítico/genética , Proteínas de Plantas/metabolismo , Piruvato Ortofosfato Diquinase/metabolismo , Zea mays/enzimologia , Substituição de Aminoácidos/genética , Especificidade de Anticorpos , Sítios de Ligação/genética , Catálise , Histidina/metabolismo , Mutagênese Sítio-Dirigida , Fosforilação , Proteínas de Plantas/classificação , Proteínas de Plantas/genética , Piruvato Ortofosfato Diquinase/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Treonina/metabolismo
2.
FEBS Lett ; 413(1): 169-73, 1997 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-9287137

RESUMO

A key regulatory enzyme of the C4-photosynthetic pathway is stromal pyruvate,orthophosphate dikinase (PPDK, EC 2.7.9.1). As a pivotal enzyme in the C4 pathway, it undergoes diurnal light-dark regulation of activity which is mediated by a single bifunctional regulatory protein (RP). RP specifically inactivates PPDK in the dark by an ADP-dependent phosphorylation of an active-site Thr residue (Thr-456 in maize). Conversely, RP activates inactive PPDK in the light by phosphorolytic dephosphorylation of this target Thr-P residue. We have employed a His-tagged maize recombinant C4 PPDK for directed mutagenesis of this active-site regulatory Thr. Three such mutants (T456V, T456S, T456D) were analyzed with respect to overall catalysis and regulation by exogenous maize RP. Substitution with Val and Ser at this position does not affect overall catalysis, whereas Asp abolishes enzyme activity. With respect to regulation by RP, it was found that Ser can effectively substitute for the wild-type Thr residue in that mutant enzyme is phosphorylated and inactivated by RP. The T456V mutant, however, could not be phosphorylated and was, thus, resistant to ADP-dependent inactivation by RP.


Assuntos
Piruvato Ortofosfato Diquinase/metabolismo , Zea mays/enzimologia , Difosfato de Adenosina/farmacologia , Ácido Aspártico/genética , Ácido Aspártico/fisiologia , Histidina/genética , Mutagênese Sítio-Dirigida , Fosforilação , Piruvato Ortofosfato Diquinase/genética , Serina/genética , Serina/fisiologia , Treonina/fisiologia , Valina/genética , Valina/fisiologia , Zea mays/genética
3.
Photosynth Res ; 49(1): 83-9, 1996 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24271536

RESUMO

The gene for C4-pyruvate,orthophosphate dikinase (PPDK) from maize (Zea mays) was cloned into an Escherichia coli expression vector and recombinant PPDK produced in E. coli cells. Recombinant enzyme was found to be expressed in high amounts (5.3 U purified enzyme-activity liter(-1) of induced cells) as a predominantly soluble and active protein. Biochemical analysis of partially purified recombinant PPDK showed this enzyme to be equivalent to enzyme extracted from illuminated maize leaves with respect to (i) molecular mass, (ii) specific activity, (iii) substrate requirements, and (iv) phosphorylation/inactivation by its bifunctional regulatory protein.

4.
J Bacteriol ; 172(9): 5044-51, 1990 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-2118506

RESUMO

A DNA-binding factor (VF1) partially purified from Anabaena sp. strain PCC 7120 vegetative cell extracts by heparin-Sepharose chromatography was found to have affinity for the xisA upstream region. The xisA gene is required for excision of an 11-kilobase element from the nifD gene during heterocyst differentiation. Previous studies of the xisA upstream sequences demonstrated that deletion of this region is required for the expression of xisA from heterologous promoters in vegetative cells. Mobility shift assays with a labeled 250-base-pair fragment containing the binding sites revealed three distinct DNA-protein complexes. Competition experiments showed that VF1 also bound to the upstream sequences of the rbcL and glnA genes, but the rbcL and glnA fragments showed only single complexes in mobility shift assays. The upstream region of the nifH gene formed a weak complex with VF1. DNase footprinting and deletion analysis of the xisA binding site mapped the binding to a 66-base-pair region containing three repeats of the consensus recognition sequence ACATT.


Assuntos
Cianobactérias/genética , Proteínas de Ligação a DNA/metabolismo , Regiões Promotoras Genéticas , Sequência de Bases , Cianobactérias/metabolismo , Desoxirribonuclease I , Dados de Sequência Molecular , Plasmídeos , Mapeamento por Restrição , Homologia de Sequência do Ácido Nucleico
5.
J Bacteriol ; 172(7): 3925-31, 1990 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-2113913

RESUMO

An 11-kilobase-pair element interrupts the nifD gene in vegetative cells of Anabaena sp. strain PCC 7120. The nifD element normally excises only from the chromosomes of cells that differentiate into nitrogen-fixing heterocysts. The xisA gene contained within the element is required for the excision. Shuttle vectors containing the Escherichia coli tac consensus promoter fused to various 5' deletions of the xisA gene were constructed and conjugated into Anabaena sp. strain PCC 7120 cells. Some of the expression plasmids resulted in excision of the nifD element in a high proportion of vegetative cells. Excision of the element required deletion of an xisA 5' regulatory region which presumably blocks expression in Anabaena sp. strain PCC 7120 vegetative cells but not in E. coli. Strains lacking the nifD element grew normally in medium containing a source of combined nitrogen and showed normal growth and heterocyst development in medium lacking combined nitrogen. The xisA gene was shown to be the only Anabaena gene required for the proper rearrangement in E. coli of a plasmid containing the borders of the nifD element.


Assuntos
Cianobactérias/genética , Expressão Gênica , Genes Bacterianos , Fixação de Nitrogênio/genética , Regiões Promotoras Genéticas , Southern Blotting , Deleção Cromossômica , Clonagem Molecular/métodos , DNA/genética , DNA/isolamento & purificação , Escherichia coli/genética , Rearranjo Gênico , Plasmídeos , Mapeamento por Restrição , Transcrição Gênica
6.
Planta ; 179(1): 81-8, 1989 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24201425

RESUMO

The assimilation of (14)CO2 into the C4 acids malate and aspartate by leaves of C3, C4 and C3-C4 intermediate Flaveria species was investigated near the CO2 compensation concentration Γ(*) in order to determine the potential role of phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) in reducing photorespiration in the intermediates. Relative to air concentrations of CO2, the proportion of CO2 fixed by PEP carboxylase at Γ(*) increased in all six C3-C4 intermediate species examined. However, F. floridana J.R. Johnston and F. ramosissima Klatt were shown to be markedly less responsive to reduced external CO2, with only about a 1.6-fold enhancement of CO2 assimilation by PEP carboxylase, as compared to a 3.0- to 3.7-fold increase for the other C3-C4 species examined, namely, F. linearis Lag., F. anomala B.L. Robinson, F. chloraefolia A. Gray and F. pubescens Rydb. The C3 species F. pringlei Gandoger and F. cronquistii A.M. Powell exhibited a 1.5- and 2.9-fold increase in labeled malate and aspartate, respectively, at Γ(*). Assimilation of CO2 by PEP carboxylase in the C4 species F. trinervia (Spreng.) C. Mohr, F. australasica Hook., and the C4-like species F. brownii A.M. Powell was relatively insensitive to subatmospheric levels of CO2. The interspecific variation among the intermediate Flaverias may signify that F. floridana and F. ramosissima possess a more C4-like compartmentation of PEP carboxylase and ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) between the mesophyll and bundle-sheath cells. Chasing recently labeled malate and aspartate with (12)CO2 for 5 min at Γ(*) resulted in an apparent turnover of 25% and 30% of the radiocarbon in these C4 acids for F. ramosissima and F. floridana, respectively. No substantial turnover was detected for F. linearis, F. anomala, F. chloraefolia or F. pubescens. With the exception of F. floridana and F. ramosissima, it is unlikely that enhanced CO2 fixation by PEP carboxylase at the CO2 compensation concentration is a major mechanism for reducing photorespiration in the intermediate Flaveria species. Moreover, these findings support previous related (14)CO2-labeling studies at air-levels of CO2 which indicated that F. floridana and F. ramosissima were more C4-like intermediate species. This is further substantiated by the demonstration that F. floridana PEP carboxylase, like the enzyme in C4 plants, undergoes a substantial activation (2.2-fold) upon illuminating dark-adapted green leaves. In contrast, light activation was not observed for the enzyme in F. linearis or F. chloraefolia.

7.
Proc Natl Acad Sci U S A ; 85(13): 4696-9, 1988 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-3133659

RESUMO

The Chlamydomonas reinhardtii chloroplast mutant 68-4PP is phenotypically indistinguishable from wild type at 25 degrees C but fails to grow photosynthetically at 35 degrees C. It had about 30% of the wild-type level of ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) holoenzyme and carboxylase activity when grown at 25 degrees C, but less than 15% when grown at 35 degrees C. Pulse-labeling with 35S showed that the decrease in enzyme level at the restrictive temperature was not a result of reduced synthesis of enzyme subunits. The CO2/O2 specificity factor (VCKO/VOKC, where VC and VO are Vmax values for carboxylation and oxygenation and KC and KO are Km values for CO2 and O2) of the mutant enzyme was found to be significantly less than that of the wild-type enzyme (54 +/- 2 and 62 +/- 1, respectively), and this alteration was accompanied by increases in KO and KC and a decrease in VC/VO. DNA sequencing revealed a single missense mutation in the 68-4PP chloroplast large-subunit gene. This mutation causes leucine to be replaced by phenylalanine at position 290 in the large-subunit polypeptide sequence. These results (i) support previous studies that implicated this region of the large subunit as an important structural component of the enzyme's function and (ii) demonstrate that chloroplast genetic modification of the CO2/O2 specificity factor of a plant-type carboxylase/oxygenase is feasible.


Assuntos
Dióxido de Carbono/metabolismo , Chlamydomonas/genética , Oxigênio/metabolismo , Proteínas de Plantas/genética , Ribulose-Bifosfato Carboxilase/genética , Chlamydomonas/enzimologia , Cloroplastos/enzimologia , Proteínas de Plantas/metabolismo , Ribulose-Bifosfato Carboxilase/metabolismo
8.
Planta ; 173(3): 411-8, 1988 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24226549

RESUMO

Comparative (14)CO2 pulse-(12)CO2 chase studies performed at CO2 compensation (Γ)-versus air-concentrations of CO2 demonstrated a four-to eightfold increase in assimilation of (14)CO2 into the C4 acids malate and aspartate by leaves of the C3-C4 intermediate species Panicum milioides Nees ex Trin., P. decipiens Nees ex Trin., Moricandia arvensis (L.) DC., and M. spinosa Pomel at Γ. Specifically, the distribution of (14)C in malate and aspartate following a 10-s pulse with (14)CO2 increases from 2% to 17% (P. milioides) and 4% to 16% (M. arvensis) when leaves are illuminated at the CO2 compensation concentration (20 µl CO2/l, 21% O2) versus air (340 µl CO2/l, 21% O2). Chasing recently incorporated (14)C for up to 5 min with (12)CO2 failed to show any substantial turnover of label in the C4 acids or in carbon-4 of malate. The C4-acid labeling patterns of leaves of the closely related C3 species, P. laxum Sw. and M. moricandioides (Boiss.) Heywood, were found to be relatively unresponsive to changes in pCO2 from air to Γ. These data demonstrate that the C3-C4 intermediate species of Panicum and Moricandia possess an inherently greater capacity for CO2 assimilation via phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) at the CO2 compensation concentration than closely related C3 species. However, even at Γ, CO2 fixation by PEP carboxylase is minor compared to that via ribulosebisphosphate carboxylase (EC 4.1.1.39) and the C3 cycle, and it is, therefore, unlikely to contribute in a major way to the mechanism(s) facilitating reduced photorespiration in the C3-C4 intermediate species of Panicum and Moricandia.

9.
Plant Cell Rep ; 7(4): 266-9, 1988 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24241763

RESUMO

A heterotrophic cotton (Gossypium hirsutum L. cv. Stoneville 825) cell suspension culture was adapted to grow photoautotrophically. After two years in continuous photoautotrophic culture at 5% CO2 (balance air), the maximum growth rate of the photoautotrophic cell line was a 400% fresh weight increase in eight days. The Chl concentration was approximately 500 µg per g fresh weight.Elevated CO2 (1%-5%) was required for culture growth, while the ambient air of the culture room (600 to 700 ul CO2 1(-1)) or darkness were lethal. The cell line had no net photosynthesis at 350 ul 1(-1) CO2, 2% O2, and dark respiration ranged from 29 to 44 µmol CO2 mg(-1) Chl h(-1). Photosynthesis was inhibited by O2. The approximate 1:1 ratio of ribulose 1,5-bisphosphate carboxylase (RuBPcase) to phosphoenolpyruvate carboxylase (PEPcase) (normally about 6:1 in mature leaves of C3 plants) was due to low RuBPcase activity relative to that of C3 leaves, not to high PEPcase activity. The PEPcase activity per unit Chl in the cell line was identical to that of spinach leaves, while the RuBPcase activity was only 15% of the spinach leaf RuBPcase activity. RuBPcase activity in the photoautotrophic cells was not limited by a lack of activation in vivo, since the enzyme in a rapidly prepared cell extract was 73% activated. No evidence of enzyme inactivation by secondary compounds in the cells was found as can be found with cotton leaves. Low RuBPcase activity and high respiration rates are most likely important factors in the low photosynthetic efficiency of the cells at ambient CO2.

10.
Plant Physiol ; 77(4): 851-6, 1985 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16664149

RESUMO

Leaf photosynthesis and ribulose bisphosphate carboxylase activation level were inhibited in several mutants of the C(3) crucifer Arabidopsis thaliana which possess lesions in the photorespiratory pathway. This inhibition occurred when leaves were illuminated under a photorespiratory atmosphere (50% O(2), 350 microliters per liter CO(2), balance N(2)), but not in nonphotorespiratory conditions (2% O(2), 350 microliters per liter CO(2), balance N(2)). Inhibition of carboxylase activation level was observed in strains with deficient glycine decarboxylase, serine transhydroxymethylase, serine-glyoxylate aminotransferase, glutamate synthase, and chloroplast dicarboxylate transport activities, but inhibition did not occur in a glycolate-P phosphatase-deficient strain. Also, the photorespiration inhibitor aminoacetonitrile produced a decline in leaf and protoplast ribulose bisphosphate carboxylase activation level, but was without effect on intact chloroplasts. Fructose bisphosphatase, a light-activated enzyme which is strongly dependent on stromal pH and Mg(2+) for regulation, was unaffected by conditions which caused inhibition of ribulose bisphosphate carboxylase. Thus, the mechanism of inhibition does not appear to involve changes in stromal Mg(2+) and pH but rather is associated with metabolite flux through the photorespiratory pathway.

11.
Curr Genet ; 9(1): 83-9, 1984 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24173514

RESUMO

The rcl-u-1-18-5B chloroplast mutation results in the absence of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) holoenzyme in the green alga Chlamydomonas reinhardii. The 18-5B mutant strain lacks photosynthesis and displays alight-sensitive, acetate-requiring phenotype. In the present investigations, revertants of 18-5B were recovered that regained photosynthetic competence. These revertants have decreased levels of Rubisco holoenzyme relative to wild type and display heteroplasmicity, segregating wild-type (revertant) and acetate-requiring phenotypes during vegetative growth or through meiosis. One of these revertants, R10-I, was studied further. The heteroplasmicity associated with photoautotrophically-grown R10-I was found to be stable through subcloning and heritable through several crosses. During growth in acetate medium in the dark, where photosynthesis provides no selective advantage, the wild-type phenotype was lost. Acetate-requiring segregants became homoplasmic but wild-type segregants did not. Organellar intergenic-suppression is discussed in light of the observed stable heteroplasmicity.

12.
Plant Physiol ; 68(4): 981-2, 1981 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16662038

RESUMO

Tripropyltin restores medium acidification by washed corn root tissue in which electrogenic H(+) efflux has been blocked by ATPase inhibitors or injury. However, the restored H(+) efflux is not electrogenic and will not drive K(+) influx, and, by itself, tripropyltin is inhibitory to K(+) influx. Tripropyltin elicits a 5-fold increase in endogenous chloride efflux, and Cl(-)/OH(-) exchange can, thus, account for the observed acidification of the medium. This explanation cannot be applied equally to the acidification produced by the K(+)/H(+) exchanging ionophore nigericin.

13.
Plant Physiol ; 68(1): 107-10, 1981 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16661850

RESUMO

The mechanism of cytokinin-induced cell expansion in cotyledons excised from dark-grown seedlings of radish (Raphanus sativus L.) and cucumber (Cucumus sativus L.) was studied. Cotyledons were incubated in dim light with or without 17 micromolar zeatin for periods up to 3 days. Fresh weights and osmotic potentials were measured daily. Cell wall extensibility properties were measured before and after the growth period. Also, experiments in which radish cotyledons were grown in mannitol solutions of various concentrations were performed. Comparisons of growth rates and increases of tissue osmotic potentials (toward zero) during growth without mannitol indicate that wall extensibility increased during the growth period and that this extensibility was enhanced by zeatin.Extensibility values derived from growth rates in mannitol provided indirect evidence of zeatin-increased wall extensibility. These conclusions were verified by direct measurements of plasticity with an Instron extensiometer. Thus, growth stimulation of excised cotyledons by cytokinins apparently involves wall loosening, in addition to previously demonstrated increases of K(+) absorption and formation of reducing sugars.

14.
Plant Physiol ; 67(4): 832-5, 1981 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16661763

RESUMO

The block in the electrogenic H(+) efflux produced by protein synthesis inhibitors in corn root tissue can be released or by-passed by addition of fusicoccin or nigericin. The inhibition also lowers cell potential, and the release repolarizes. Associated with the inhibition of H(+) efflux is inhibition of K(+) influx and the growth of the root tip; fusicoccin partially relieves these inhibitions, but nigericin does not. The inhibition of H(+) efflux which arises from blocking the proton channel of the ATPase by oligomycin or N,N'-dicyclohexylcarbodiimide can also be partially relieved by fusicoccin, but not by nigericin; the inhibition produced by diethylstilbestrol is not relieved by fusicoccin.The results are discussed in terms of the presumed mode of action of fusicoccin on the plasmalemma ATPase. Inhibition of protein synthesis appears to inactivate the proton channel of the ATPase, possibly as the indirect result of disrupted metabolism. Fusicoccin reactivates or bypasses the blocked channel.

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