Purification and Molecular and Catalytic Properties of Phosphoribulokinase from the Cyanobacterium Chlorogloeopsis fritschii

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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confidence: 99%

Chlamydomonas reinhardtii Phosphoribulokinase

Roesler¹,

Ogren²

1990

“…Antiserum against PRK purified from Chlorogloeopsisfritschii (17) was raised in rabbits as described previously ( 18). Null serum was collected by bleeding the rabbit prior to the injection of PRK.…”

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confidence: 99%

“…Antiserum prepared against the large subunit ofRuBisCO from tobacco (15) (13,18). PRK activity was measured by coupling the formation of RuBP to the subsequent incorporation of Na-H'4C03 into acid-stable material in the presence of excess exogenous RuBisCO as described previously (17). In tests to determine the effects of C. fritschii PRK antiserum on the activity of the C. paradoxa enzyme, 200 ,g of antiserum or null serum were incubated with the soluble and particulate cell-free extracts for 15 min at 30'C before assaying.…”

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confidence: 99%

Immunocytochemical Localization of Phosphoribulose Kinase in the Cyanelles of Cyanophora paradoxa and Glaucocystis nostochinearum

Mangeney

Hawthornthwaite²,

Codd³

et al. 1987

ABSTRACUThe distribution of phosphoribulose kinase (PRK) in the cyanelies of Cyawphora paradoxa Korschikoff and Glucocystis xostochinearwm Itzigsohn was studied by protein A-gold immunoelectron microscopy. In both endocyanomes, antiserum against PRK heavily labeled the thylakoid region of the cyanelles, whereas little or no label was present over the carboxysomes. Antiserum aginst ribulose 1,5-bisphosphate carboxyase/ oxygenase by contrast heavily labeled the carboxysomes of each endocyanome. In vitro studies of PRK distribution in cell-free extacts of C. pardoxa showed that 93% of the enzyme was in the soluble fractin Quantitative immunoelectron mcroscopy showed that more than 99% of the PRK in the cyanelle of C. parwdoxa was lalized in the thylakoid region. We conclude that the carboxysomes of cyanelles like the carboxysomes of autotrophic prokaryotes and the pyrenoids of green algl chloroplasts do not contain phosphoribulose kinase.

“…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

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.