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 pH and kinetic studies of chloroplast sedoheptulose-1,7-bisphosphatase from spinach (Spinacia oleracea)

1988 ◽  
Vol 253 (1) ◽  
pp. 249-254 ◽  
Author(s):  
F Cadet ◽  
J C Meunier
Keyword(s):  
Spinacia Oleracea ◽  
Ph Dependence ◽  
Kinetic Studies ◽  
Competitive Inhibitor ◽  
Kinetic Properties ◽  
Enzyme Specificity ◽  
Active Centre ◽  
Specificity Constant ◽  
The One ◽  
Fructose 1,6 Bisphosphate

The aim of this paper is to study some steady-state kinetic properties of sedoheptulose-1,7-bisphosphatase, its pH-dependence and the effect of a substrate analogue, fructose 2,6-bisphosphate. Studies were carried out with sedoheptulose 1,7-bisphosphate and with fructose 1,6-bisphosphate, an alternative substrate. The pK values are identical for both substrates, and fructose 2,6-bisphosphate behaves like a competitive inhibitor. These results suggest that there exists a unique active site for either sedoheptulose 1,7-bisphosphate or fructose 1,6-bisphosphate on the enzyme molecule. Increasing Mg2+ concentrations shifted the optimum pH. As for fructose-1,6-bisphosphatase, we believe that this shift is due to the neutralization of negative charges near the active centre [Cadet, Meunier & Ferté (1987) Eur. J. Biochem. 162, 393-398]. The free species of sedoheptulose 1,7-bisphosphate and fructose 1,6-bisphosphate are not the usual substrates of enzyme, nor is Mg2+. But the kinetics relative to the (Mg2+-substrate4-)2- complex is not consistent with this complex being the substrate. An explanation of this discrepancy is proposed, involving both the negative charges near the active centre and the positive charges of Mg2+. The observed Vmax. of the reduced enzyme is 65% of the theoretical Vmax. for both substrates, but the observed Vmax. relative to sedoheptulose 1,7-bisphosphate is 3 times the one relative to fructose 1,6-bisphosphate. The specificity constant (kcat./Km), 1.62 × 10(6) M-1.s-1 with respect to sedoheptulose 1,7-bisphosphate compared with 5.5 × 10(4) M-1.s-1 with respect to fructose 1,6-bisphosphate, indicates that the enzyme specificity towards sedoheptulose 1,7-bisphosphate is high but not absolute.

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

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 Effects of phosphorylation on the kinetic properties of rat liver fructose-1,6-bisphosphatase

1984 ◽  
Vol 222 (1) ◽  
pp. 125-130 ◽  
Author(s):  
D W Meek ◽  
H G Nimmo
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Rat Liver ◽  
Kinetic Properties ◽  
Purification Procedure ◽  
Dependent Protein Kinase ◽  
Fructose 1,6 Bisphosphatase ◽  
Dependent Protein ◽  
A Subunit ◽  
Fructose 1,6 Bisphosphate

A new purification procedure for rat liver fructose-1,6-bisphosphatase that involves use of Procion Red-Sepharose is described. The purified enzyme was homogeneous, had a subunit Mr of 40 000-41 000 and seemed to be undegraded. The enzyme could be phosphorylated by cyclic AMP-dependent protein kinase with a stoicheiometry of one per subunit. Phosphorylation caused a 2-fold decrease in the Km of the enzyme for fructose 1,6-bisphosphate, but did not affect its allosteric responses to AMP, Mg2+ and fructose 2,6-bisphosphate.

scholarly journals Purification and properties of arylsulphatase B of human liver

1976 ◽  
Vol 153 (2) ◽  
pp. 415-421 ◽  
Author(s):  
S I O Agogbua ◽  
C H Wynn
Keyword(s):  
Active Site ◽  
Human Liver ◽  
Specific Activity ◽  
Cold Treatment ◽  
Apparent Molecular Weight ◽  
Acid Group ◽  
Kinetic Properties ◽  
Amino Acid Group ◽  
Purification And Properties ◽  
Purification Scheme

1. A purification scheme for an arylsulphatase B from human liver is described. Specificity of purification was achieved by the use of the affinity chromatography on an agrose-4-hydroxy-2-nitrophenyl sulphate derivative. The scheme provides a rapid and convenient method for preparation of a highly purified enzyme. 2. The purified enzyme was examined by isoelectric focusing electrophoresis on polyacrylamide gel and by ultracentrifugation and was found to be catalytically homogenous, with an apparent molecular weight of 50000 and a specific activity of 93.3 units/mg of protein. 3. The kinetic properties of the purified preparation and the effect of various amino acid group-specific reagents on the catalysis of the enzyme are described. The involvement of histidine residues in the active site of the enzyme is suggested. 4. The purified enzyme lost activity rapidly on freezing. The implication of this observation is discussed in terms of a possible dissociation-reaggregation phenomenon induced by cold treatment.

scholarly journals Phosphofructokinases A and B from Mycobacterium tuberculosis Display Different Catalytic Properties and Allosteric Regulation

2021 ◽  
Vol 22 (3) ◽  
pp. 1483
Author(s):  
Jan Snášel ◽  
Iva Machová ◽  
Veronika Šolínová ◽  
Václav Kašička ◽  
Marcela Krečmerová ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Biochemical Characterization ◽  
Metabolic Enzyme ◽  
Kinetic Properties ◽  
Reaction Products ◽  
Hypoxic Conditions ◽  
Glycolytic Activity ◽  
Gene And Protein Expression ◽  
Fructose 1,6 Bisphosphatase ◽  
Fructose 1,6 Bisphosphate

Tuberculosis (TB) remains one of the major health concerns worldwide. Mycobacterium tuberculosis (Mtb), the causative agent of TB, can flexibly change its metabolic processes during different life stages. Regulation of key metabolic enzyme activities by intracellular conditions, allosteric inhibition or feedback control can effectively contribute to Mtb survival under different conditions. Phosphofructokinase (Pfk) is one of the key enzymes regulating glycolysis. Mtb encodes two Pfk isoenzymes, Pfk A/Rv3010c and Pfk B/Rv2029c, which are differently expressed upon transition to the hypoxia-induced non-replicating state of the bacteria. While pfkB gene and protein expression are upregulated under hypoxic conditions, Pfk A levels decrease. Here, we present biochemical characterization of both Pfk isoenzymes, revealing that Pfk A and Pfk B display different kinetic properties. Although the glycolytic activity of Pfk A is higher than that of Pfk B, it is markedly inhibited by an excess of both substrates (fructose-6-phosphate and ATP), reaction products (fructose-1,6-bisphosphate and ADP) and common metabolic allosteric regulators. In contrast, synthesis of fructose-1,6-bisphosphatase catalyzed by Pfk B is not regulated by higher levels of substrates, and metabolites. Importantly, we found that only Pfk B can catalyze the reverse gluconeogenic reaction. Pfk B thus can support glycolysis under conditions inhibiting Pfk A function.

scholarly journals Influence of rapid changes in cytosolic pH on oxidative phosphorylation in skeletal muscle: theoretical studies

2002 ◽  
Vol 365 (1) ◽  
pp. 249-258 ◽  
Author(s):  
Bernard KORZENIEWSKI ◽  
Jerzy A. ZOLADZ
Keyword(s):  
Skeletal Muscle ◽  
Creatine Kinase ◽  
Oxidative Phosphorylation ◽  
Kinetic Properties ◽  
Main Role ◽  
Anaerobic Glycolysis ◽  
Cytosolic Ph ◽  
Intensive Exercise ◽  
Proton Production ◽  
The Stability

Cytosolic pH in skeletal muscle may vary significantly because of proton production/consumption by creatine kinase and/or proton production by anaerobic glycolysis. A computer model of oxidative phosphorylation in intact skeletal muscle developed previously was used to study the kinetic effect of these variations on the oxidative phosphorylation system. Two kinds of influence were analysed: (i) via the change in pH across the inner mitochondrial membrane and (ii) via the shift in the equilibrium of the creatine kinase-catalysed reaction. Our simulations suggest that cytosolic pH has essentially no impact on the steady-state fluxes and most metabolite concentrations. On the other hand, rapid acidification/alkalization of cytosol causes a transient decrease/increase in the respiration rate. Furthermore, changes in pH seem to affect significantly the kinetic properties of transition between resting state and active state. An increase in pH brought about by proton consumption by creatine kinase at the onset of exercise lengthens the transition time. At intensive exercise levels this pH increase could lead to loss of the stability of the system, if not compensated by glycolytic H+ production. Thus our theoretical results stress the importance of processes/mechanisms that buffer/compensate for changes in cytosolic proton concentration. In particular, we suggest that the second main role of anaerobic glycolysis, apart from additional ATP supply, may be maintaining the stability of the system at intensive exercise.

Purification to homogeneity and characterization of nonproteolyzed potato (Solanum tuberosum) tuber hexokinase 1

Botany ◽  
2011 ◽  
Vol 89 (5) ◽  
pp. 289-299 ◽  
Author(s):  
Marie-Claude Moisan ◽  
Jean Rivoal
Keyword(s):  
Solanum Tuberosum ◽  
Specific Activity ◽  
Solanum Tuberosum L ◽  
Extraction Procedure ◽  
Hydrophobic Interaction Chromatography ◽  
Solanum Chacoense ◽  
Kinetic Properties ◽  
Chromatographic Separations ◽  
The Stability ◽  
Fast Flow

We have developed an extraction procedure that improves the stability of potato ( Solanum tuberosum L.) tuber hexokinase (HK) after extraction. Using this protocol, we showed that at least four HK isoforms are present in this tissue, and they can be separated by hydrophobic-interaction chromatography on a butyl-Sepharose™ 4 Fast Flow column. One of the main HK isoforms was purified to homogeneity using further chromatographic separations on red dye, DEAE Fractogel, hydroxyapatite, cibacron blue, and MonoQ matrices. HK-specific activity of this fraction (10.2 U·mg protein–1) corresponds to an enrichment of more than 5500-fold, with a yield of 0.9%. This is the highest reported HK-specific activity from a plant source. The purified enzyme consisted of a monomer with a subunit apparent Mr of 51 kDa when analyzed by SDS–PAGE. This polypeptide was recognized by affinity-purified anti- Solanum chacoense Bitt. recombinant HK IgGs. The protein was digested with trypsin and its digestion products were subjected to MS – MS sequencing after HPLC separation. The sequences of these tryptic peptides matched the predicted coding sequence of the S. tuberosum HK1 gene with a coverage of 57%. Examination of the kinetic properties of the purified protein HK1 indicates that it may be regulated by the internal O2 concentration of the tuber because of its sensitivity to acidic pHs and inhibition by ADP.

scholarly journals Concomitant polymorphs of aryl-ether amine via catemer and dimer carboxylic acid supramolecular interactions and their effect on optical properties

2021 ◽  
Author(s):  
Mehboobali Pannipara ◽  
Abdullah G Al-Sehemi
Keyword(s):  
Optical Properties ◽  
Carboxylic Acid ◽  
Crystal Lattice ◽  
Functional Groups ◽  
Molecular Orbital ◽  
Acid Group ◽  
Supramolecular Interactions ◽  
Aryl Ether ◽  
Stability Calculation ◽  
The Stability

Abstract Carboxylic acid supramolecular synthon exhibited dimer or catemer motifs in the crystal lattice depend on the substituent and other functional groups present in the structure. In general, presence of other competing functional groups produced catemer motifs whereas unsubstituted acids showed dimer. In this manuscript, we have synthesized a new aryl ether amine-based Schiff base with carboxylic acid functionality ( 1 ) and demonstrated polymorphic structure via catemer ( 1a ) and dimer ( 1b ) motifs in the solid state. In both the structure, carboxylic acid group adopted different orientation in the crystal lattice. The different H-bonding lead to modulation of optical properties that was further supported highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) calculation. Further the stability calculation indicates that catemer structure was more stable by 8.54 kcal/mole relative to dimer motifs. In contrast, naphthyl group attached carboxylic acid structure did not show neither dimer nor catemer motifs in the crystal lattice as compared to diethylaminophenyl group, which confirm the presence of other substituent or competing functional groups strongly influence on the motifs of supramolecular interactions.

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