N-terminal truncation affects the kinetics and structure of fructose-6-phosphate 2-kinase/fructose-2,6-bisphosphatase from Arabidopsis thaliana

2001 ◽  
Vol 359 (3) ◽  
pp. 591-597 ◽  
Author(s):  
Dorthe VILLADSEN ◽  
Tom H. NIELSEN
Keyword(s):  
Arabidopsis Thaliana ◽  
Gel Filtration ◽  
Full Length ◽  
Dihydroxyacetone Phosphate ◽  
Kinetic Properties ◽  
N Terminus ◽  
Fructose 1,6 Bisphosphate ◽  
Substrate Affinities ◽  
Allosteric Properties

The enzyme fructose-6-phosphate 2-kinase (F6P,2K; 6-phosphofructo-2-kinase)/fructose-2,6-bisphosphatase(F26BPase) catalyses the formation and degradation of the regulatory metabolite fructose 2,6-bisphosphate. A cDNA encoding the bifunctional plant enzyme isolated from Arabidopsis thaliana (AtF2KP) was expressed in yeast, and the substrate affinities and allosteric properties of the affinity-purified enzyme were characterized. In addition to the known regulators 3-phosphoglycerate, dihydroxyacetone phosphate, fructose 6-phosphate and Pi, several metabolites were identified as important new effectors. PPi, phosphoenolpyruvate and 2-phosphoglycerate strongly inhibited F6P,2K activity, whereas fructose 1,6-bisphosphate and 6-phosphogluconate inhibited F26BPase activity. Furthermore, pyruvate was an activator of F6P,2K and an inhibitor of F26BPase. Both kinase and phosphatase activities were rapidly inactivated by mild heat treatment (42°C, 10min), but the presence of phosphate protected both enzyme activities from inactivation. In addition to the catalytic regions, the Arabidopsis enzyme comprises a 345-amino-acid N-terminus of unknown function. The role of this region was examined by the expression of a series of N-terminally truncated enzymes. The full-length and truncated enzymes were analysed by gel-filtration chromatography. The full-length enzyme was eluted as a homotetramer, whereas the truncated enzymes were eluted as monomers. Deletion of the N-terminus decreased the kinase/phosphatase activity ratio by 4-fold, and decreased the affinity for the substrate fructose 6-phosphate. The data show that the N-terminus is important both for subunit assembly and for defining the kinetic properties of the enzyme.

Molecular and kinetic characterisation of sugarcane pyrophosphate: fructose-6-phosphate 1-phosphotransferase and its possible role in the sucrose accumulation phenotype

2007 ◽  
Vol 34 (6) ◽  
pp. 517 ◽  
Author(s):  
Jan-Hendrik Groenewald ◽  
Frederik Coenraad Botha
Keyword(s):  
Specific Activity ◽  
Gel Filtration ◽  
Kinetic Properties ◽  
Sucrose Accumulation ◽  
Forward Reaction ◽  
Ph Optimum ◽  
Aggregation State ◽  
Inorganic Pyrophosphate ◽  
Fructose 1,6 Bisphosphate

The amount of pyrophosphate: fructose-6-phosphate 1-phosphotransferase (PFP) activity in sugarcane internodal tissue is inversely correlated with sucrose content. To help elucidate this apparent role of PFP in sucrose accumulation in sugarcane we have determined its molecular and kinetic properties. Sugarcane PFP was purified 285-fold to a final specific activity of 4.23 µmol min–1 mg–1 protein. It contained two polypeptides of 63.2 and 58.0 kDa respectively, at near equal amounts that cross-reacted with potato PFP-α and –β antiserum. In gel filtration analyses the native enzyme eluted in three peaks of 129, 245 and 511 kDa, corresponding to dimeric, tetrameric and octameric forms, respectively and fructose 2,6-bisphosphate (Fru 2,6-P2) influenced this aggregation state. Both the glycolytic (forward) and gluconeogenic (reverse) reactions had relative broad pH optima between pH 6.7 and 8.0. The Fru 2,6-P2 saturation curves were hyperbolic with approximate Ka values of 69 and 82 nm for the forward and reverse reactions, respectively. The enzyme showed hyperbolic saturation curves for all its substrates with Km values comparable with that of other plant PFP, i.e. 150, 37, 39 and 460 µM for fructose 6-phosphate, inorganic pyrophosphate, fructose 1,6-bisphosphate and inorganic phosphate, respectively. Sugarcane PFP’s molecular and kinetic characteristics differed slightly from that of other plant PFP in that: (i) Fru 2,6-P2 directly induced the octameric state from the dimeric state; (ii) Fru 2,6-P2 shifted the pH optimum for the forward reaction to a slightly more basic pH; and (iii) Fru 2,6-P2 increased the Vmax for the forward and reverse reactions by similar amounts.

Comparison of full-length and conserved segments of wheat dehydrin DHN-5 overexpressed in Arabidopsis thaliana showed different responses to abiotic and biotic stress

2016 ◽  
Vol 43 (11) ◽  
pp. 1048 ◽  
Author(s):  
Marwa Drira ◽  
Moez Hanin ◽  
Khaled Masmoudi ◽  
Faiçal Brini
Keyword(s):  
Arabidopsis Thaliana ◽  
Fungal Infections ◽  
Alternaria Solani ◽  
Triticum Aestivum L ◽  
Full Length ◽  
Plant Tolerance ◽  
Wild Type ◽  
Biotic And Abiotic Stresses ◽  
Related Stress

Dehydrins (DHNs) are among the most common proteins accumulated in plants under water-related stress. They typically contain at least three conserved sequences designated as the Y-, S- and K-segments. The present work aims to highlight the role of the K-segments in plant tolerance to biotic and abiotic stresses. For this purpose, transgenic Arabidopsis thaliana (L.) Heyhn. lines expressing distinct wheat (Triticum aestivum L.) DHN-5 truncated constructs with or without the K-segments were generated. Our results showed that unlike the derivative lacking a K-segment, constructs containing only one or two K-segments enhanced the tolerance of A. thaliana to diverse stresses and were similar to the full-length wheat DHN-5. Moreover, compared with the wild-type and the YS form, the transgenic plants overexpressing wheat DHN-5 with K-segments maintained higher superoxide dismutase, catalase and peroxide dismutase enzymatic activity, and accumulated lower levels of H2O2 and malondialdehyde. In addition, we demonstrated that lines like A. thaliana overexpressing wheat DHN-5 showed increased resistance to fungal infections caused by Botrytis cinerea and Alternaria solani. Finally, the overexpression of the different forms of wheat DHN-5 led to the regulation of the expression of several genes involved in the jasmonic acid signalling pathway.

scholarly journals Elucidation of the role of glutamine synthetase seed isoform GLN1;5 in Arabidopsis thaliana (L.) with a reverse genetics approach

2019 ◽  
Vol 71 (3) ◽  
pp. 443-453
Author(s):  
Milan Dragicevic ◽  
Katarina Cukovic ◽  
Snezana Zdravkovic-Korac ◽  
Ana Simonovic ◽  
Milica Bogdanovic ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Glutamine Synthetase ◽  
Reverse Genetics ◽  
Technological Development ◽  
Expression Patterns ◽  
Dry Weight ◽  
Grain Filling ◽  
Kinetic Properties ◽  
Plant Nitrogen

Glutamine synthetase (E.C. 6.3.1.2) is a key enzyme of plant nitrogen metabolism that assimilates ammonia into glutamine. The Arabidopsis thaliana genome encodes one chloroplastic (GLN2) and five cytosolic (GLN1;1 ? GLN1;5) isoforms with different expression patterns, kinetic properties, regulation and functions. Physiological roles of different isoforms have been elucidated mainly by studying knockout mutants. However, the role of GLN1;5, which is expressed in dry seeds, remains unknown. To clarifty the function of GLN1;5, we studied a GLN1;5 knockout line (GLN1;5KO) homozygous for T-DNA insertion within the GLN1;5. GLN1;5 deficiency results in a phenotype with slightly delayed bolting and fewer siliques. The dry weight of GLN1;5KO seeds was 73.3% of wild-type (WT) seed weight, with seed length 90.9% of WT seeds. Finally, only 18.33% of the mutant seeds germinated in water within 10 days in comparison to 34.67% of WT seeds. KNO3 strongly stimulated germination of both GLN1;5KO and WT seeds, while germination in the presence of increasing NH4Cl concentrations potentiated the differences between the two genotypes. It can be concluded that GLN1;5 activity supports silique development and grain filling and that it has a role in ammonium reassimilation in the seed, as well as assimilation and/or detoxification of ammonium from the environment. [Project of the Serbian Ministry of Education, Science and Technological Development, Grant no. ON173024 and Grant no. ON173015]

scholarly journals Distinct domain requirements for EAP45 in HIV budding, late endosomal recruitment, and cytokinesis

2020 ◽  
Author(s):  
Bo Meng ◽  
Pedro P. Vallejo Ramirez ◽  
Katharina M. Scherer ◽  
Ezra Bruggeman ◽  
Julia C. Kenyon ◽  
...  
Keyword(s):  
Lipid Membranes ◽  
Biological Process ◽  
Full Length ◽  
Live Cells ◽  
Fluorescent Labelling ◽  
Endosomal Membrane ◽  
Cellular Processes ◽  
N Terminus ◽  
Main Component

The scission of lipid membranes is a common biological process, often mediated by ESCRT complexes in concert with VPS4 assembling around the separation point. The functions of the ESCRT-I and ESCRT-III complexes are well established in certain of these cellular processes; however, the role of ESCRT-II remains contentious. Here, we devised a SNAP-tag fluorescent labelling strategy to understand the domain requirements of EAP45, the main component of ESCRT-II, in HIV egress, late endosome recruitment, and cytokinesis. We used TIRF microscopy to measure the spatial co-occurrence of the HIV structural polyprotein Gag with full length EAP45 in both fixed and live cells. Gag colocalises with the full length EAP45 comparably to ALIX, but this is lost on deletion of the EAP45 N terminus. Our findings reveal the H0 domain of the EAP45 protein is essential for linking to ESCRT-I during HIV budding and in anchoring at the late endosomal membrane, however in cytokinesis it is the Glue domain that is critical.

scholarly journals Control of the fructose 6-phosphate/fructose 1,6-bisphosphate cycle in isolated hepatocytes by glucose and glucagon. Role of a low-molecular-weight stimulator of phosphofructokinase

1980 ◽  
Vol 192 (3) ◽  
pp. 887-895 ◽  
Author(s):  
Emile Van Schaftingen ◽  
Louis Hue ◽  
Henri-Géry Hers
Keyword(s):  
Molecular Weight ◽  
Incubation Medium ◽  
Liver Extract ◽  
Gel Filtration ◽  
Isolated Hepatocytes ◽  
Low Molecular Weight ◽  
Experimental Conditions ◽  
Fructose 1,6 Bisphosphate ◽  
Acid Labile

1. Recycling of metabolites between fructose 6-phosphate and triose phosphates has been investigated in isolated hepatocytes by the randomization of carbon between C(1) and C(6) of glucose formed from [1-14C]galactose. 2. Randomization of carbon atoms was regularly observed with hepatocytes isolated from fed rats and was then little influenced by the concentration of glucose in the incubation medium. It was decreased by about 50% in the presence of glucagon. 3. Randomization of carbon atoms by hepatocytes isolated from starved rats was barely detectable at physiological concentrations of glucose in the incubation medium, but was greatly increased with increasing glucose concentrations. It was nearly completely suppressed by glucagon. These large changes can be attributed to parallel variations in the activity of phosphofructokinase. 4. The main factors that appear to control the activity of phosphofructokinase under these experimental conditions are the concentration of fructose 6-phosphate, the concentration of fructose 1,6-bisphosphate and also the affinity of the enzyme for fructose 6-phosphate. 5. The affinity of phosphofructokinase for fructose 6-phosphate was diminished by incubation of the cells in the presence of glucagon and also by filtration of an extract of hepatocytes through Sephadex G-25 and by purification of the enzyme. When assayed at 0.25 or 0.5mm-fructose 6-phosphate, the activity of phosphofructokinase present in a liver Sephadex filtrate was increased by a low-molecular-weight effector, which could be isolated from a liver extract by ultrafiltration, gel filtration or heat treatment, but was rapidly destroyed in trichloroacetic acid, even in the cold. This effector appears to be a highly acid-labile phosphoric ester. Its concentration was greatly increased in hepatocytes incubated in the presence of glucose and was decreased in the presence of glucagon.

scholarly journals A subunit interface mutant of yeast pyruvate kinase requires the allosteric activator fructose 1,6-bisphosphate for activity

1995 ◽  
Vol 310 (1) ◽  
pp. 117-123 ◽  
Author(s):  
R A Collins ◽  
T McNally ◽  
L A Fothergill-Gilmore ◽  
H Muirhead
Keyword(s):  
Pyruvate Kinase ◽  
Mutant Enzyme ◽  
Hill Coefficient ◽  
Kinetic Properties ◽  
Variant Form ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Kinase Gene ◽  
Fructose 1,6 Bisphosphate ◽  
Allosteric Properties

A variant form of yeast pyruvate kinase (EC 2.7.1.40) with Ser-384 mutated to proline has been engineered in order to study the allosteric properties of this enzyme. Both the mutant and wild-type enzymes were overexpressed in a strain of yeast in which the genomic copy of the pyruvate kinase gene had been disrupted by an insertion of the Ura3 gene. Both enzymes were purified to homogeneity and their kinetic properties characterized. The wild-type enzyme displays sigmoid kinetics with respect to phosphoenolpyruvate (PEP) concentration, and is activated by the allosteric effect fructose 1,6-bisphosphate with concomitant reduction in co-operativity. In contrast, the mutant was found to be dependent on the presence of the effector for catalytic activity and was inactive in its absence. The fully activated mutant enzyme had a kcat. 1.6 times greater than that of the wild-type enzyme. The mutation introduced into the enzyme is in an intersubunit contact which is known to be critical for the allosteric properties of the enzyme, and is far removed from the active site. The major effect of the mutation seems to be to stabilize the low-affinity T state of the apoenzyme, although kcat. is also affected. The S0.5 for PEP and S0.5 for ADP of the wild-type enzyme were 0.22 +/- 0.004 and 0.15 +/- 0.01 mM respectively (means +/- S.E.M.). In the activated mutant enzyme, these kinetic parameters increased to 0.67 +/- 0.03 and 0.43 +/- 0.03 mM respectively. The cooperativity between ADP-binding sites was altered in the mutant enzyme, with the Hill coefficient (h) for ADP increasing to 1.65 +/- 0.07 in the presence of the effector, compared with a value of 0.01 +/- 0.07 for the wild-type enzyme under the same conditions. CD spectroscopy revealed the secondary structure of the mutant enzyme to be little different from that of the wild-type enzyme, indicating that the two enzymes have similar secondary structures in solution. Precise tertiary and quaternary structures such as intersubunit and interdomain interactions may be modified. An improved purification procedure has been devised that allows large quantities of enzyme to be rapidly prepared.

scholarly journals Identity and Kinetic Properties of a Protein (ACTIN) which Binds Metabolic Platelet ADP in an Ethanol-Insoluble Form

1977 ◽  
Author(s):  
J.L. Daniel ◽  
L. Robkin ◽  
H. Holmsen
Keyword(s):  
Gel Filtration ◽  
Kinetic Properties ◽  
Kinetic Behavior ◽  
Insoluble Form ◽  
Metabolic Pool ◽  
Rapid Equilibrium ◽  
The Individual ◽  
Nucleotide Binding Proteins ◽  
Platelet Functions

Thirty to fifty percent of the metabolic ADP in platelets is bound to protein and the complex is insoluble in ethanol, from which it can be isolated from platelets and subsequently extracted by perchloric acid. We have tentatively identified the protein as actin based on the following : 1) comparison of the amount of ethanol insoluble ADP and the actin content of the platelets and other nucleotide-binding proteins are not present in sufficient amounts; 2) its behavior on a gel filtration column; 3) its solubility properties; 4) the exchangeability of the bound ADP in platelet lysates. The kinetic behavior of the “actin”-bound nucleotide in theintactplatelet was studies in cells that had their (ethanol-soluble and-insoluble) metabolic pool preequilibrated with 14C-adenine. The change with time of the 3H/14C ratio in the pools after addition ofH-adenine was measured. In the resting platelet the 3H/14C ratio increased at the same rate in the ethanol soluble and-insoluble pool, indicating rapid equilibrium between the pools.However, when 3H-adenine was added to 14C-labelled platelets that had been incubated with antimycin and deoxyglucose, the 3H/14C ratio in the protein bound pool clearly lagged behind that of the soluble pool, the 3H/14C in the individual nucleotides suggested that soluble ATP was a precursor for protein-bound ADP. These results indicate that ATP is concumed to maintain a certain equilibrium between G-and F-actin in resting platelets. This technique 3H/14C ratio of studying the kinetic platelets parameters of “actin”-bound ADP may be of use for elucidating the role of contractile proteins in platelet functions.

The role of SERT N-terminus in amphetamine-induced efflux

2016 ◽  
Vol 4 (Suppl. 3) ◽  
pp. A4.17
Author(s):  
Fatma Aslı Erdem
Keyword(s):  
N Terminus
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