Sedoheptulose-1,7-bisphosphate phosphatase activity of chloroplast fructose-1,6-bisphosphatase: identification of enzymes hydrolysing fructose-1,6-bisphosphate and sedoheptulose-1,7-bisphosphate in stromal extracts from chloroplasts of spinach (Spinaci

1998 ◽  
Vol 25 (5) ◽  
pp. 531 ◽  
Author(s):  
Anthony R. Ashton
Keyword(s):  
Phosphatase Activity ◽  
Spinacia Oleracea ◽  
Reduced Form ◽  
Fructose 1,6 Bisphosphatase ◽  
Fructose 1,6 Bisphosphate ◽  
Hydrolysis Of ◽  
Enzyme Species

The identity of enzymes present in soluble extracts of spinach(Spinacia oleracea) chloroplasts that are capable ofhydrolysing fructose-1,6-bisphosphate and sedoheptulose-1,7-bisphosphate hasbeen investigated using antibodies against purified spinach chloroplastfructose-1,6-bisphosphatase (EC 3.1.3.11). The activity of purifiedfructose-1,6-bisphosphatase, which can exist in a less active oxidised form ora more active reduced form as well as total fructose-1,6-bisphosphatase instromal extracts is inhibited completely by the antiserum. Apparently, only asingle enzyme, which can exist in an oxidised or reduced form, is responsiblefor hydrolysis of fructose-1,6-bisphosphate in the chloroplast. Purifiedchloroplast fructose-1,6-bisphosphatase can also exhibitsedoheptulose-1,7-bisphosphatase activity, but only when reduced. Oxidisedchloroplast stromal extracts contain little or nosedoheptulose-1,7-bisphosphatase activity whereas reduced extracts containsedoheptulose-1,7-bisphosphatase activity. Antiserum against fructose-1,6-bisphosphatase does not inhibit sedoheptulose-1,7-bisphosphatase activitydetectable at pH 8 or less with 2 mM Mg2+ butsubstantially inhibits (up to 60%) the sedoheptulose-1,7-bisphosphataseactivity at higher pH or Mg2+ concentration, i.e.conditions under which the chloroplast fructose-1,6-bisphosphatase exhibitssedoheptulose-1,7-bisphosphatase activity. Apparently, the chloroplast stromacontains at least two enzyme species capable of hydrolysingsedoheptulose-1,7-bisphosphate, a specific sedoheptulose-1,7-bispho-sphatase(EC 3.1.3.37) and the chloroplast fructose-1,6-bisphosphatase.

scholarly journals Properties of oxidized and reduced spinach (Spinacia oleracea) chloroplast fructose-1,6-bisphosphatase activated by various agents

1991 ◽  
Vol 278 (3) ◽  
pp. 787-791 ◽  
Author(s):  
T Chardot ◽  
J C Meunier
Keyword(s):  
Spinacia Oleracea ◽  
Acid Group ◽  
Lag Phase ◽  
Kinetic Properties ◽  
Amino Acid Group ◽  
Progress Curve ◽  
Fructose 1,6 Bisphosphatase ◽  
Specificity Constant ◽  
The Stability ◽  
Fructose 1,6 Bisphosphate

Fructose-1,6-bisphosphatase (FBPase) can be reduced and activated by either dithiothreitol or reduced thioredoxin. This activation is pH-dependent. An amino acid group with a pK value of 5.55 is involved in this process. Both enzyme forms can also be stimulated by agents such as fructose 1,6-bisphosphate, Mg2+, Ca2+ and Ca2+/fructose 1,6-bisphosphate. FBPase reduced by dithiothreitol is more strongly activated than the enzyme reduced by thioredoxin. The specificity constant (kcat./Km) is enhanced over 2.5-25-fold and 1.5-2-fold (depending on the agent used) for FBPase reduced by dithiothreitol and thioredoxin respectively. In both cases, no new kinetic properties appeared. The pH-activity profile of the stimulated enzyme is slightly shifted towards the acidic side with respect to the reduced enzyme. A lag phase is observed in the progress curve of both enzymic forms, treated or untreated. Each agent used to stimulate must induce a new conformation of the enzyme, more active than the initial one, characterized by a specificity constant and a relaxation time. This lag phase tends to disappear when the assay temperature is increased. Temperature has the same effect on the activity of oxidized, reduced and stimulated FBPase, but different effects on the stability of the different forms.

scholarly journals NADP(H) Phosphatase Activities of Archaeal Inositol Monophosphatase and Eubacterial 3′-Phosphoadenosine 5′-Phosphate Phosphatase

2007 ◽  
Vol 73 (17) ◽  
pp. 5447-5452 ◽  
Author(s):  
Chikako Fukuda ◽  
Shigeyuki Kawai ◽  
Kousaku Murata
Keyword(s):  
Phosphatase Activity ◽  
Physiological Role ◽  
Inositol Polyphosphate ◽  
Nad Kinase ◽  
Inositol Monophosphatase ◽  
Tertiary Structures ◽  
E Coli ◽  
Fructose 1,6 Bisphosphatase ◽  
Fructose 1,6 Bisphosphate

ABSTRACT NADP(H) phosphatase has not been identified in eubacteria and eukaryotes. In archaea, MJ0917 of hyperthermophilic Methanococcus jannaschii is a fusion protein comprising NAD kinase and an inositol monophosphatase homologue that exhibits high NADP(H) phosphatase activity (S. Kawai, C. Fukuda, T. Mukai, and K. Murata, J. Biol. Chem. 280:39200-39207, 2005). In this study, we showed that the other archaeal inositol monophosphatases, MJ0109 of M. jannaschii and AF2372 of hyperthermophilic Archaeoglobus fulgidus, exhibit NADP(H) phosphatase activity in addition to the already-known inositol monophosphatase and fructose-1,6-bisphosphatase activities. Kinetic values for NADP+ and NADPH of MJ0109 and AF2372 were comparable to those for inositol monophosphate and fructose-1,6-bisphosphate. This implies that the physiological role of the two enzymes is that of an NADP(H) phosphatase. Further, the two enzymes showed inositol polyphosphate 1-phosphatase activity but not 3′-phosphoadenosine 5′-phosphate phosphatase activity. The inositol polyphosphate 1-phosphatase activity of archaeal inositol monophosphatase was considered to be compatible with the similar tertiary structures of inositol monophosphatase, fructose-1,6-bisphosphatase, inositol polyphosphate 1-phosphatase, and 3′-phosphoadenosine 5′-phosphate phosphatase. Based on this fact, we found that 3′-phosphoadenosine 5′-phosphate phosphatase (CysQ) of Escherichia coli exhibited NADP(H) phosphatase and fructose-1,6-bisphosphatase activities, although inositol monophosphatase (SuhB) and fructose-1,6-bisphosphatase (Fbp) of E. coli did not exhibit any NADP(H) phosphatase activity. However, the kinetic values of CysQ and the known phenotype of the cysQ mutant indicated that CysQ functions physiologically as 3′-phosphoadenosine 5′-phosphate phosphatase rather than as NADP(H) phosphatase.

scholarly journals Coxiella burnetii inhibits host immunity by a protein phosphatase adapted from glycolysis

2021 ◽  
Vol 119 (1) ◽  
pp. e2110877119
Author(s):  
Yong Zhang ◽  
Jiaqi Fu ◽  
Shuxin Liu ◽  
Lidong Wang ◽  
Jiazhang Qiu ◽  
...  
Keyword(s):  
Phosphatase Activity ◽  
Coxiella Burnetii ◽  
Protein Phosphatase ◽  
Bacterial Pathogen ◽  
Sugar Metabolism ◽  
Host Immunity ◽  
Host Cells ◽  
Functional Screening ◽  
Type Iv ◽  
Fructose 1,6 Bisphosphate

Coxiella burnetii is a bacterial pathogen that replicates within host cells by establishing a membrane-bound niche called the Coxiella-containing vacuole. Biogenesis of this compartment requires effectors of its Dot/Icm type IV secretion system. A large cohort of such effectors has been identified, but the function of most of them remain elusive. Here, by a cell-based functional screening, we identified the effector Cbu0513 (designated as CinF) as an inhibitor of NF-κB signaling. CinF is highly similar to a fructose-1,6-bisphosphate (FBP) aldolase/phosphatase present in diverse bacteria. Further study reveals that unlike its ortholog from Sulfolobus tokodaii, CinF does not exhibit FBP phosphatase activity. Instead, it functions as a protein phosphatase that specifically dephosphorylates and stabilizes IκBα. The IκBα phosphatase activity is essential for the role of CinF in C. burnetii virulence. Our results establish that C. burnetii utilizes a protein adapted from sugar metabolism to subvert host immunity.

A phenoxo-bridged dicopper(ii) complex as a model for phosphatase activity: mechanistic insights from a combined experimental and computational study

2017 ◽  
Vol 46 (12) ◽  
pp. 4038-4054 ◽  
Author(s):  
Suman K. Barman ◽  
Totan Mondal ◽  
Debasis Koley ◽  
Francesc Lloret ◽  
Rabindranath Mukherjee
Keyword(s):  
Dft Calculations ◽  
Phosphatase Activity ◽  
Theoretical Approach ◽  
Computational Study ◽  
Hydrolysis Of

Hydrolysis of RNA-model substrate HPNP by a dicopper(ii) complex has been studied with combined experimental and theoretical approach. Involvement of H-bonding has been probed by DFT calculations.

New Reactive Coenzyme Analogues for Affinity Labeling of NAD+ and NADP+ Dependent Dehydrogenases

1995 ◽  
Vol 50 (7-8) ◽  
pp. 476-486
Author(s):  
Reinhard Jeck ◽  
Michael Scholze ◽  
Anja Tischlich ◽  
Christoph Woenckhaus ◽  
Jürgen Zimmermann
Keyword(s):  
Adenosine Diphosphate ◽  
Competitive Inhibitor ◽  
Hydrogen Donor ◽  
Reduced Form ◽  
Irreversible Inhibitor ◽  
Hydrocarbon Chains ◽  
Phosphoric Acid Ester ◽  
Acetyl Group ◽  
Hydrolysis Of ◽  
Phosphate Groups

Abstract Reactive coenzyme analogues ω-(3-diazoniumpyridinium)alkyl adenosine diphosphate were prepared by reaction of ω-(3-aminopyridinium)alkyl adenosine diphosphate with nit­rous acid. In these compounds the nicotinamide ribose is substituted by hydrocarbon chains of varied lengths (n-ethyl to n-pentyl). The diazonium compounds are very unstable and decompose rapidly at room temperature. They show a better stability at 0 °C. L actate and alcohol dehydrogenase do not react with any of the analogues. Glyceraldehyde-3-phosphate dehydrogenase reacts rapidly with the diazonium pentyl compound. Decreasing the length of the alkyl chain significantly decreases the inactivation velocity. 3α,20β-Hydroxysteroid dehydrogenase reacts at 0 °C with the ethyl homologue and slowly with the propyl compound. The butyl-and pentyl analogues do not inactivate at 0 °C. Tests with 14C -labeled 2-(3-diazoniumpyridinium)ethyl adenosine diphosphate show that complete loss of enzyme activity results after incorporation of 2 moles of inactivator into 1 mole of tetrameric enzyme. 4-(3-Acetylpyridinium)butyl 2 ′-phospho-adenosine diphosphate, a structural analogue of NADP +, was prepared by condensation of adenosine-2,3-cyclophospho-5′-phosphomorpholidate with (3-acetylpyridinium)butyl phosphate, followed by hydrolysis of the cyclic phosphoric acid ester with 2 ′:3′-cyclonucleotide-3′-phosphodiesterase. Because of the redox potential (-315 mV) and the distance between the pyridinium and phosphate groups, this analogue is a hydrogen acceptor and its reduced form a hydrogen donor in tests with alcohol dehyd roge­nase from Thermoanaerobium brockii. The reduced form of the coenzyme analogue also is a hydrogen donor with glutathione reductase. With other NADP +-dependent dehydrogenases the com pound has been show n to be a competitive inhibitor against the natural coenzyme. The acetyl group reacts with bromine to form the bromoacetyl group. This reactive bromoacetyl analogue is a specific active-site directed irreversible inhibitor of isocitrate dehydrogenase.

scholarly journals Phosphomonoesterase hydrolysis of polyphosphoinositides in rat kidney: Properties and subcellular localization of the enzyme system

1975 ◽  
Vol 150 (3) ◽  
pp. 537-551 ◽  
Author(s):  
P H Cooper ◽  
J N Hawthorne
Keyword(s):  
Phosphatase Activity ◽  
Metal Ion ◽  
Rat Kidney ◽  
Gradient Centrifugation ◽  
Sodium Deoxycholate ◽  
Subcellular Fractionation ◽  
Kidney Cortex ◽  
Ph Optimum ◽  
Triton X ◽  
Hydrolysis Of

Tthe properties of diphosphoinositide and triphosphoinositide phosphatases from rat kidney homogenate were studied in an assay system in which non-specific phosphatase activity was eliminated. The enzymes were not completely metal-ion dependent and were activated by Mg2+. The detergent sodium deoxycholate, Triton X-100 and Cutscum inhibited the reaction; cetyltrimethylammonium bromide only activated when added with the subtrates and in the presence Mg2+. Both enzymes had a pH optimum of 7.5. Ca2+ and Li+ both activated triphosphoinositide phosphatase, but Ca2+ inhibited and L+ had little effect on diphosphoinositide phosphatase. Cyclic AMP had no effect on either enzyme. The enzymes were three times more active in kidney cortex than in the medulla. On subcellular fractionation of kidney-cortex homogenates by differential and density-gradient centrifugation, the distribution of the enzymes resembled that of thiamin pyrophosphatase (assayed in the absence of ATP), suggesting localization in the Golgi complex. However, the distribution differed from that of the liver Golgimarker galactosyltransferase. Activities of both diphosphoinositide and triphosphoinositide phosphatases and thiamin pyrophosphatase were low in purified brush-border fragments. Further experiments indicate that at least part of the phosphatase activity is soluble.

Regulation of Fructose 1,6-Bisphosphatase Activity of Chlorella by Mole Mass Change

1996 ◽  
Vol 51 (9-10) ◽  
pp. 639-645 ◽  
Author(s):  
N. Grotjohann
Keyword(s):  
Enzyme Regulation ◽  
Fold Increase ◽  
Substrate Affinity ◽  
Kinetic Properties ◽  
Enzyme Form ◽  
Phosphorylated Compounds ◽  
Fructose 1,6 Bisphosphatase ◽  
Chloroplast Stroma ◽  
Fructose 1,6 Bisphosphate

Fast protein liquid chromatography on Superose 6 of partially purified FBPase II from Chlorella reveals a 1350 kDa-form at pH 6.0 and a 67 kDa-form at pH 8.5. Treatment of the large enzyme form with 5mᴍ concentrations of Mg2+, F1,6P2, DTT or ATP leads to dissociation into smaller ones of 215 -470 kDa. Aggregation/dissoziation is a reversible process, as has been shown for the effect of F1,6P2 and of pH, by rechromatography. The change in mole mass results in alterations of the activitiy and of the kinetic properties of the enzyme forms, obtained. Dissociation results in a 4 - 6 fold increase in activity, as can be shown for F1,6P2-treated samples. Halfsaturation constants, as well as the degree of cooperativity of the 67- and the 1350- kDa form, are different for substrate affinity, activation by Mg2+ and DTT, and for inhibition by ATP. Both enzyme forms hydrolyse fructose 1,6 bisphosphate and seduheptulose 1,7 bisphosphate better than other phosphorylated compounds. The ratio of F1,6P2- to SDP-cleavage is 100:58 for the small enzyme form and 100: 84 for the large one. Activation of FBPase II in the light and inactivation in the dark is discussed on the basis of different oligomeric forms of the enzyme, generated by changes in the concentration of intermediates and effectors in the chloroplast stroma, leading to dissociation or aggregation. The conclusion is drawn that oligomerization of key enzymes, resulting in enzyme forms with different activities and different kinetic properties, might provide an effective mechanism for enzyme regulation in vivo

scholarly journals A Comparison of Human Leukocyte Phosphatase Activity toward Sodium β-Glycerophosphate, Adenosine 5'-Phosphate and Glucose 1-Phosphate

Blood ◽  
1959 ◽  
Vol 14 (4) ◽  
pp. 415-422 ◽  
Author(s):  
JAMES H. FOLLETTE ◽  
WILLIAM N. VALENTINE ◽  
JOHN REYNOLDS
Keyword(s):  
Alkaline Phosphatase ◽  
Phosphatase Activity ◽  
Cell Population ◽  
Alkaline Phosphatase Activity ◽  
Human Leukocyte ◽  
Maximal Activity ◽  
Phosphate Esters ◽  
Chronic Myelocytic Leukemia ◽  
Myelocytic Leukemia ◽  
Hydrolysis Of

Abstract The ability of human leukocyte enzymes to hydrolyze phosphorus is compared in terms of the conventional substrate sodium β-glycerophosphate and the metabolically important phosphate esters, adenosine 5'-phosphate and glucose 1-phosphate. At pH 9.9, there is marked and comparable variation in phosphatase activity toward all three substrates, this being low in chronic myelocytic leukemia and high in the presence of infection and certain "stressful" states. Moreover, substrate mixture experiments show no increased hydrolysis of phosphorus when two substrates are present in the incubation mixture. Increased phosphatase activity toward both glucose 1-phosphate and sodium β-glycerophosphate resulted when corticosteroids were administered in large doses for 72 hours. The data, while not providing absolute proof, are compatible with the hydrolysis of phosphorus at pH 9.9, being due in the case of all three substrates to the activity of the same phosphomonoesterase or group of phosphomonesterases. At pH 5.5, phosphatase activity toward both sodium β-glycerophosphate and adenosine 5'-phosphate was likewise demonstrated, but, in leukocytes, the pH of maximal activity varies from subject to subject and is dependent to a large extent on the amount of the highly variable "alkaline phosphatase" activity present in any given cell population at the time of analysis.

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