Electrophoretic and chromatographic separation of two fructose-1,6-bisphosphatase forms from Synechococcus leopoliensix

Gerbling, Klaus-Peter; Steup, Martin; Latzko, Erwin

doi:10.1007/bf00414449

Cited by 14 publications

(7 citation statements)

References 15 publications

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“…The occurrence of FBP isozymes is widespread, underscoring the important physiological role of this enzyrme (1,8,27,28). In organisms that utilize the Calvin cycle, FBP must regulate the flow of carbon from CO2 assimilation to other metabolic pathways in addition to its gluconeogenic function.…”

Section: Resultsmentioning

confidence: 99%

Localization and mapping of CO2 fixation genes within two gene clusters in Rhodobacter sphaeroides

Gibson

Tabita

1988

J Bacteriol

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Two fructose 1,6-bisphosphatase structural genes (fbpA and fbpB) have been identified within two unlinked gene clusters that were previously shown to contain the Rhodobacter sphaeroides sequences that code for form I and form II ribulose 1,5-bisphosphate carboxylase-oxygenase and phosphoribulokinase. The fbpA and fbpB genes were localized to a region immediately upstream from the corresponding prk4 and prkB sequences and were found to be transcribed in the same direction as the phosphoribulokinase and ribulose 1,5-bisphosphate carboxylase-oxygenase genes based on inducible expression of fructose 1,6-bisphosphatase activity directed by the lac promoter. A recombinant plasmid was constructed that contained the tandem JbpA and prkA genes inserted downstream from the lac promoter in plasmid pUC18. Both gene products were expressed in Escherichia coli upon induction of transcription with isopropyl P-D-thiogalactoside, demonstrating that the two genes can be cotranscribed. A Zymomonas mobils glyceraldehyde 3-phosphate-dehydrogenase gene (gap) hybridized to a DNA sequence located approximately 1 kilobase upstream from the form H ribulose 1,5-bisphosphate carboxylase-oxygenase gene. Although no corresponding gap sequence was found within the form I gene cluster, an additional region of homology was detected immediately upstream from the sequences that encode the form I and form H ribulose 1,5-bisphosphate carboxylase-oxygenases.

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Section: Resultsmentioning

confidence: 99%

Localization and mapping of CO2 fixation genes within two gene clusters in Rhodobacter sphaeroides

Gibson

Tabita

1988

J Bacteriol

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“…In recent investigations on fructose bisphosphatase from a photoautotrophic procaryote, Synechococcus leopoliensis (formerly Anacvstis nidulans), two proteins exhibiting bisphosphatase activity have been resolved by electrophoretic techniques (9). Form B, which probably represents the major proportion of the total extractable fructose bisphosphatase activity, has been purified to homogeneity and characterized (10).…”

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confidence: 99%

“…Second, FBP (plus Mg2l) is also the substrate (10 (FRG). Axenic cultures were grown as previously described (9).…”

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confidence: 99%

Fructose 1,6-Bisphosphatase Form B from Synechococcus leopoliensis Hydrolyzes both Fructose and Sedoheptulose Bisphosphate

Gerbling¹,

Steup²,

Latzko³

1986

Plant Physiol.

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ABSTRACITThe substrate specificity of purified fructose bisphosphatase form B from Synechococcus leopoliensis (EC 3.1.3.11; cf. K-P Gerbling, M Steup, E Latzko 1985 Eur J Biochem 147: 207-215) has been investigated. Of the phosphate esters tested only fructose 1,6-bisphosphate and sedoheptulose 1,7-bisphosphate were hydrolyzed by the enzyme. Both sugar bisphosphates were cleaved at the carbon 1-ester. Fructose-and sedoheptulose bisphosphate stabilized the activated (i.e. tetrameric) state of the enzyme and prevented a slow inactivation that is observed in the absence of sugar bisphosphates. With the activated enzyme, kinetic constants (half-saturating substrate concentrations, maximal reaction velocity, and the catalytical constant) were similar for both fructose-and sedoheptulose bisphosphate. The data suggest that fructose bisphosphatase form B from Synechococcus leopoliensis can catalyze both bisphosphatase reactions within the reductive pentose phosphate cycle.In the reductive pentose phosphate cycle two sugar bisphosphate esters, FBP' and sedoheptulose 1,7-bisphosphate, are cleaved specifically at the carbon 1-ester. Both phosphatase reactions probably play an important role in regulating the flux through the entire cycle (12, 17; but see 7). However, conflicting data have been reported on the enzymology of both phosphatase activities. In some photo-and chemoautotrophic cells, fructose and sedoheptulose bisphosphatase activities have been attributed to distinct enzymes specific either for sedoheptulose bisphosphate or FBP (13,20). In contrast, in other organisms fructose bisphosphatase has been shown to hydrolyze sedoheptulose bisphosphate as well (1,2,18) and the existence of a sedoheptulose bisphosphate specific phosphatase has been questioned (1 1).

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“…More recent studies of Synechococcus leopoliensis have shown two electrophoretically distinct FBPases in this cyanobacterium (11,12). The most prevalent form was shown to undergo DTT activation and a dimer-tetramer interconversion similar to the spinach photosynthetic enzyme (12).…”

Section: Discussionmentioning

confidence: 98%

“…However, the phycobiont of P. rufescens is a cyanobacterium (1,11,25). The FBPases isolated from these cyanobacteria appear to be similar to the photosynthetic enzyme in other respects (1,22,25 (Tables I, III, IV).…”

Section: Discussionmentioning

confidence: 99%

Isolation and Characterization of Two Enzymes Capable of Hydrolyzing Fructose-1,6-Bisphosphatase from the Lichen Peltigera rufescens

Brown

Kershaw²

1986

Plant Physiol.

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Two enzymes capable of hydrolyzing fructose-1,6-bisphosphate (FBP) have been isolated from the foliose lichen Peltigera rufescens (Weis) Mudd. These enzymes can be separated using Sephadex G-100 and DEAE Sephacel chromatography. One enzyme has a pH optimum of 6.5, and a substrate affinity of 228 micromolar FBP. This enzyme does not require MgCI2 for activity, and is inhibited by AMP. The second enzyme has a pH optimum of 9.0, with no activity below pH 7.5. This enzyme responds sigmoidally to Mg2", with half-saturation concentration of 2.0 millimolar MgCI2, and demonstrates hyperbolic kinetics for FBP (K. = 39 micromolar). This enzyme is activated by 20 millimolar dithiothreitol, is inhibited by AMP, but is not affected by fructose-2-6-bisphosphate. It is hypothesized that the latter enzyme is involved in the photosynthetic process, while the former enzyme is a nonspecific acid phosphatase.FBPase2 (EC 3.1.3.11) is an important enzyme of carbon metabolism, involved in gluconeogenesis, the pentose phosphate cycle and photosynthesis. As a result, three forms of this enzyme are usually present in most plants (15,17,28), distinguishable by their compartmentation, regulatory properties and their response to differing pH and ionic environments. One FBPase acts as a Mg2+-independent acid phosphatase (17,20), while the other two are Mg2+-dependant alkaline phosphatases. Ofthe latter two, the cytosolic FBPase is inhibited by AMP, F-2,6-P, and high concentrations of FBP (8, 13,30). The photosynthetic enzyme is not inhibited by these compounds, but it is activated by thioredoxin or DTT (4,5,23,29 2-mercaptoethanol, and 20 mm sodium metabisulfite, adjusted to pH 6.6) (10 ml g-' dry weight) and insoluble PVP (0.5 g-g-' dry weight) were added to the drained thalli and this was ground with a mortar and pestle. The slurry was further processed for 20 min with a motorized tissue homogenizer. The resulting suspension was sonicated for a total of 20 min, and then centrifuged in a swinging bucket rotor on a Beckman TJ 6 refrigerated centrifuge for 20 min at 1500g. The supernatant was reserved and the pellet was resuspended in approximately 300 ml of the extraction buffer, then resonicated and again centrifuged. The supernatants were combined and the final pellet discarded.FBPase Purification. The enzyme was purified using a modification of the method of Buchanan et al. (4). The crude extract was acidified to pH 4.5 by the addition of 1.0 M acetic acid. The precipitate formed was collected by centrifugation (I 500g for 30 min) and resuspended in 150 ml of the extraction buffer. (NH4)2SO4 was added to 40% of saturation. This solution was centrifuged in a Sorval RC 2B centrifuge at 20,000g for 15 min. The pellet was discarded and the supernatant was brought up to 80% of saturation with additional (NH4)2SO4. This was centrifuged as above, the supernatant was discarded, and the pellet was resuspended in 15 ml of extraction buffer. This was chromatographed on a 75 x 2.5 cm column ofSephadex G-100 (previously equilibrated with the...

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Electrophoretic and chromatographic separation of two fructose-1,6-bisphosphatase forms from Synechococcus leopoliensix

Cited by 14 publications

References 15 publications

Localization and mapping of CO2 fixation genes within two gene clusters in Rhodobacter sphaeroides

Localization and mapping of CO2 fixation genes within two gene clusters in Rhodobacter sphaeroides

Fructose 1,6-Bisphosphatase Form B from Synechococcus leopoliensis Hydrolyzes both Fructose and Sedoheptulose Bisphosphate

Isolation and Characterization of Two Enzymes Capable of Hydrolyzing Fructose-1,6-Bisphosphatase from the Lichen Peltigera rufescens

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