Purification and characterization of ribulose-5-phosphate kinase from spinach

Porter, Michael A.; Milanez, Sylvia; Stringer, Claude D.; Hartman, Fred C.

doi:10.1016/0003-9861(86)90185-2

Cited by 88 publications

(57 citation statements)

References 24 publications

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“…1; Table I). The specific activity of the purified Chlamydomonas phosphoribulokinase was 410 ,umol min-' mg-', similar to published values of 360 gmol min-' mg-' (12) and 410 Mmol min-' mg-' (20) for electrophoretically pure spinach phosphoribulokinase. In contrast, the purified enzyme of prokaryotic organisms had spe- (19), and 63 uM (27), to 280 gM (8) The nucleotide sequence of the Chlamydomonas phosphoribulokinase cDNA and the deduced amino acid sequence are presented in Figure 3.…”

Section: Purification and Immunoblotsupporting

confidence: 86%

“…The purification procedure developed here is similar to a procedure published for spinach phosphoribulokinase which included gel filtration, ion exchange, and affinity chromatographies, although in a different order and with different chromatographic materials (20). However, in purifying Chlamydomonas phosphoribulokinase, two aspects arose which are not a consideration in the spinach procedure.…”

Section: Discussionmentioning

confidence: 99%

“…Ammonium Common ----T-VIGLMDSGCGKSTFMRR-TS-FGG---PP-GGNPDSNTLISD- these particles, and the remainder passed through the Sephacryl column in the void volume, ahead of the phosphoribulokinase. Second, a major copurifying contaminant was present with Chlamydomonas phosphoribulokinase after the third chromatographic step, whereas the spinach enzyme was >95% pure at this stage (20). This necessitated use of the second gel filtration column which contributed to the low yield of Chlamydomonas phosphoribulokinase.…”

Section: Discussionmentioning

confidence: 99%

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Chlamydomonas reinhardtii Phosphoribulokinase

Roesler¹,

Ogren²

1990

Plant Physiol.

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The sequence and kinetic properties of phosphoribulokinase purified from Chiamydomonas reinhardtii were determined and compared with the spinach (Spinacea oleracea) enzyme. Chiamydomonas phosphoribulokinase was purified to apparent homogeneity, with a specific activity of 410 micromoles per minute per milligram. Polyclonal antibodies to the purified protein were used to isolate a Chiamydomonas cDNA clone, which, upon sequencing, was found to contain the entire coding region. (6,11,21), and its location is consistent with results of affinity labeling of the enzyme with an ATP analog (1 1). Also, the roles of several metabolic effectors in regulation of spinach phosphoribulokinase have been determined (5). Investigations of phosphoribulokinase of several prokaryotic organisms have shown that the bacterial enzyme differs markedly from the spinach enzyme with respect to kinetic parameters, subunit size and number, pH optima, activation by thiols, and regulation by metabolites (1,15,26,29).In contrast to the knowledge of the higher plant and bacterial enzymes, little biochemical characterization and no sequence data have been reported for algal phosphoribulokinase. To gain insight into structure-function relationships of this enzyme, the sequence and kinetic parameters of phosphoribulokinase from the green alga Chlamydomonas reinhardtii were determined and compared with corresponding information for the spinach enzyme. MATERIALS AND METHODS Strain and Culture ConditionsChlamydomonas reinhardtii strain 21 37A+ was maintained on an acetate medium which was described previously (28

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Section: Purification and Immunoblotsupporting

confidence: 86%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Chlamydomonas reinhardtii Phosphoribulokinase

Roesler¹,

Ogren²

1990

Plant Physiol.

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“…E-mail: jacquot@ibp.u-psud.fr Abbreviations: FBPase, fructose-1,6-bisphosphatase; DTT, dithiothreitol; NTR, NADPH thioredoxin reductase much scrutiny lately. One of the first identifications of a regulatory disulfide has been done on PRK, using chemical derivatization and site-directed mutagenesis: in this case, the regulatory site has been demonstrated to be part of the active site [4,5]. Another well-documented case is NADP-MDH.…”

Section: Introductionmentioning

confidence: 99%

Cysteine‐153 is required for redox regulation of pea chloroplast fructose‐1,6‐bisphosphatase

Jacquot¹,

et al. 1997

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Chloroplastic fructose-1,6-bisphosphatases are redox regulatory enzymes which are activated by the ferredoxin thioredoxin system via the reduction/isomerization of a critical disulfide bridge. All chloroplastic sequences contain seven cysteine residues, four of which are located in, or close to, an amino acid insertion region of approximately 17 amino acids. In order to gain more information on the nature of the regulatory site, five cysteine residues (Cys 49 , Cys 153 , Cys 173 , Cys 178 and Cys 190 ) have been modified individually into serine residues by site-directed mutagenesis. While mutations C173S and C178S strongly affected the redox regulatory properties of the enzyme, the most striking effect was observed with the C153S mutant which became permanently active and redox independent. On the other hand, the C190S mutant retained most of the properties of the wild-type enzyme (except that it could now also be partially activated by the NADPH/NTR/thioredoxin h system). Finally, the C49S mutant is essentially identical to the wild-type enzyme.

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“…PRK2 (ATP:D-ribulose 5-phosphate I-phosphotransferase, EC 2.7.1.19) is a key regulatory Calvin cycle enzyme that catalyzes the ATP-dependent phosphorylation of Ru5P to form the photosynthetic CO2 acceptor molecule RuBP: PRK Ru5P + ATP--dRuBP + ADP PRK has been purified from higher plants (6,9,10,15), cyanobacteria (13,18), a hydrogen bacterium (19), and a photosynthetic bacterium (21). The higher plant enzymes studied to date are homodimers with a native molecular mass ' of approximately 90 kD (6,9,15).…”

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confidence: 99%

Purification and Molecular and Immunological Characterization of a Unique Phosphoribulokinase from the Green Alga Selenastrum minutum

Lin

Turpin²

1992

Plant Physiol.

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A unique phosphoribulokinase (ADP:D-ribulose 5-phosphate 1-phosphotransferase, EC 2.7.1.19) has been purified to homogeneity from the green alga Selenastrum minutum. The enzyme has a native molecular mass of about 83 kilodaltons and a native isoelectric point of 5.1. The enzyme consists of two differentsized subunits of 41 and 40 kilodaltons, implying that it is a heterodimer. This is the first report of a eukaryotic heterodimeric phosphoribulokinase. The in vivo existence of two nonidentical subunits of S. minutum phosphoribulokinase was confirmed by westem blot analysis of crude protein extracts from trichloroacetic acid-killed cells. These two subunits were immunologically similar, as rabbit immunoglobulin G affinity purified against the 41 kilodalton subunit of S. minutum phosphoribulokinase (PRK) cross-reacts with the 40 kilodalton subunit and vice versa. Antibodies against S. minutum phosphoribulokinase also cross-react with the spinach enzyme. NH2-terminal sequencing revealed that the two S. minutum PRK subunits shared a considerable degree of structure homology with each other and with the enzymes from spinach and Chlamydomonas reinhardtii, but not with PRK from Rhodobacter sphaeroides. There are, however, differences between the NH2-terminal amino acid sequences of the two S. minutum PRK subunits, that imply that they are the products of separate genes or products of two different mRNAs spliced from a single gene.

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Purification and characterization of ribulose-5-phosphate kinase from spinach

Cited by 88 publications

References 24 publications

Chlamydomonas reinhardtii Phosphoribulokinase

Chlamydomonas reinhardtii Phosphoribulokinase

Cysteine‐153 is required for redox regulation of pea chloroplast fructose‐1,6‐bisphosphatase

Purification and Molecular and Immunological Characterization of a Unique Phosphoribulokinase from the Green Alga Selenastrum minutum

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