Affinity labeling and purification of spinach leaf ribulose-5-phosphate kinase

Krieger, Timothy J.; Miziorko, Henry M.

doi:10.1021/bi00360a003

Cited by 50 publications

(38 citation statements)

References 24 publications

(30 reference statements)

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“…(13,18) and lower than that reported for the spinach PRK (320-410 units/mg) (9, 10, 15) or C. reinhardtii PRK (410 units/mg) (16). The native molecular mass of S. minutum PRK, as estimated by gel filtration chromatography, was approximately 83 kD, which resembles that reported for the higher plant enzymes (6,9,15), the cyanobacterium Anabaena cylindrica enzyme (18), and the green alga S. obliquus enzyme (active form) (1 1); it is lower than that reported for the enzymes from the hydrogen bacterium A. eutrophus (19), the photosyn-41 kD 40 kD thetic bacterium R. capsulate (21), or the cyanobacterium C.fritschii (13).…”

Section: Immunological Characteristicsmentioning

confidence: 40%

“…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).…”

mentioning

confidence: 99%

“…The higher plant enzymes studied to date are homodimers with a native molecular mass ' of approximately 90 kD (6,9,15). In contrast, enzymes from the hydrogen bacterium Alcaligenes eutrophus (19), the photosynthetic bacterium Rhodopseudomonas capsulata (21), and the cyanobacterium Chlorgloeopsis fritschii (13) have their native molecular mass between 220 and 256 kD and are composed of six (13,21) or eight (19) identical subunits.…”

mentioning

confidence: 99%

“…Although less studied than the higher plant or bacterial enzyme, PRK from the green algae Scenedesmus obliquus (1 1, 12) and Chlamydomonas reinhardtii (16,17) resembles the higher plant enzyme (6,9,15) in being composed of identical subunits. Here we report the purification of PRK from the green alga Selenastrum minutum.…”

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confidence: 99%

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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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Section: Immunological Characteristicsmentioning

confidence: 40%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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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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“…Phophoribulokinase from peas was purified according to the method of Krieger and Miziorko (14) up to the step of acid precipitation. At this point the preparation was further purified by chromatography over DEAE cellulose and reactive red 120-agarose type 3000-CL (Sigma) columns as described by Porter et al (22).…”

Section: Elisamentioning

confidence: 99%

The Association of d-Ribulose- 1,5-Bisphosphate Carboxylase/Oxygenase with Phosphoribulokinase

Sainis

Merriam²,

Harris³

1989

Plant Physiol.

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When Ribulose-1,5-bisphosphate carboxylase/oxygenase was purified from spinach leaves (Spinacia oleracea) using precipitation with polyethylene glycol and MgCI2 followed by DEAE cellulose chromatography, 75% of phosphoribulokinase and 7% of phosphoriboisomerase activities copurified with ribulose-1,5-bisphosphate carboxylase/oxygenase. This enzyme preparation showed ribose-5-phosphate and ribulose-5-phosphate dependent carboxylase and oxygenase activities which were nearly equivalent to its corresponding ribulose-1,5-bisphosphate dependent activity. The ribose-5-phosphate and ribulose-5-phosphate dependent reaction rates were stable and linear for much longer time periods than the ribulose-1,5-bisphosphate dependent rates. When sucrose gradients were used to purify ribulose-1,5-bisphosphate carboxylase/oxygenase from crude stromal extracts, phosphoribulokinase was found to cosediment with ribulose-1,5-bisphosphate carboxylase. Under these conditions most of the phosphoriboisomerase activity remained with the slower sedimenting proteins. Ammonium sulfate precipitation resulted in separation of the ribulose-1,5-bisphosphate carboxylase peak from phosphonbulokinase peak. 'Abbreviations: Rubisco, ribulose-1,5-bisphosphate carboxylase/ oxygenase; Rib-5-P, ribose-5-phosphate; Ru-5-P, ribulose-5-phosphate; RuBP, ribulose-1,5-bisphosphate; fr wt, fresh weight. 368 ters such that metabolic channeling would be facilitated (25). Given the high concentration of Rubisco it is highly unlikely that Rubisco molecules would be devoid of extensive proteinprotein interactions or would be capable of free diffusion in the stromal environment. We have recently reported (23) that Rubisco, when purified from stromal extracts of pea chloroplasts on sucrose gradients, showed Rib-5-P and Ru-5-P dependent CO2 fixation activities. The expression of these activities required in addition to Rubisco either phosphoribulokinase (kinase) (Ru-5-P dependent CO2 fixation) or kinase and phosphoriboisomerase (isomerase) (Rib-5-P dependent CO2 fixation). This Rubisco preparation showed equivalent rates of CO2 fixation using RuBP or Ru-5-P when treated with 10 mM DTT. Also, the Ru-5-P dependent activity was linear for longer time periods than the RuBP dependent activity (25). These studies have suggested some binding specificity between kinase and carboxylase and some kinetic benefit to the carboxylase reaction. The work reported here is an extension of these studies using both spinach and peas. It is an effort to further characterize the association and the kinetics of this multiple reaction sequence. MATERIALS AND METHODSChloroplasts were isolated from 6 to 8 week old spinach leaves (Spinacia oleracea) and 10 to 20 d old pea shoots (Pissum sativum) according to Cerovic et al. (4). Chloroplasts were lysed in 25 mm Bicine buffer (pH 8). The membranes were removed by centrifugation (25,000g for 15 min) and supernatants were processed on sucrose gradients (5-30%) made either in 25 mM Bicine (pH 8) containing 20 mM each of MgCl2 and bicarbonate...

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Affinity labeling and purification of spinach leaf ribulose-5-phosphate kinase

Cited by 50 publications

References 24 publications

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

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

The Association of d-Ribulose- 1,5-Bisphosphate Carboxylase/Oxygenase with Phosphoribulokinase

Phosphoribulokinase: Current Perspectives on the Structure/Function Basis for Regulation and Catalysis

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