Molecular forms and thermal and kinetic properties of purified glutathione reductase from two populations of barnyard grass (Echinochloa crus-galli(L.) Beauv.: Poaceae) from contrasting climatic regions in North America

2000 ◽  
Vol 78 (7) ◽  
pp. 969-980 ◽  
Author(s):  
Nadia Hakam ◽  
Jean-Pierre Simon
Keyword(s):  
Glutathione Reductase ◽  
Thermal Adaptation ◽  
Catalytic Efficiency ◽  
Molecular Forms ◽  
Kinetic Properties ◽  
Barnyard Grass ◽  
Climatic Regions ◽  
Two Populations ◽  
Menten Constant

The thermal, kinetic, and electrophoretic properties of purified glutathione reductase (GR; EC 1.6.4.2) were analyzed in plants from two ecotypes of barnyard grass (Echinochloa crus-galli (L.) Beauv.: Poaceae) originating from sites of contrasting climates in Quebec (QUE) and Mississippi (MISS). Crude and purified GR preparations from plants of both ecotypes consisted of one homodimer isomorph with the same electrophoretic mobility in polyacrylamide gels, a similar molecular mass for the native enzyme (98 kDa) and for each subunit of the dimer (44 kDa), and an identical pI of 5.9. The electrophoretic profile of GR purified from cold-acclimated plants at 14°C light (L) : 8°C dark (D) for 10 days was similar to that of GR from plants grown at 26°C L : 20°C D. Specific activities of purified GR from QUE plants were significantly higher than those of MISS plants. In vitro GR activities from QUE and MISS plants were not differentially affected by thermodenaturation at 55 or 65°C or by cold treatments at 2°C. Apparent energies of activation (Ea) of GR purified from QUE and MISS plants were similar with the exception of estimates of Ea(oxidized glutathione) for Q10(15-5°C) for which significantly lower values were obtained for QUE plants. No differences of physiological significance were observed for Km(Michaelis-Menten constant) values of GR purified from QUE and MISS plants. However, both Vmaxand Kcat(turnover numbers) estimates were significantly higher for GR purified from QUE plants over most of the range of assay temperatures, suggesting superior catalytic efficiency for the enzyme of the cold-adapted ecotype from Québec.Key words: barnyard grass, ecotypes, electrophoresis, enzyme kinetics, glutathione reductase, thermal adaptation.

Molecular forms and kinetic properties of phosphoenolpyruvate carboxylase from barnyard grass (Echinochloa crus-galli(L.) Beauv.: Poaceae)

2000 ◽  
Vol 78 (5) ◽  
pp. 619-628
Author(s):  
Nathalie Hamel ◽  
Jean-Pierre Simon
Keyword(s):  
Phosphoenolpyruvate Carboxylase ◽  
Kinetic Property ◽  
Thermal Adaptation ◽  
Catalytic Efficiency ◽  
Molecular Forms ◽  
Kinetic Properties ◽  
Barnyard Grass ◽  
Physiological Importance ◽  
Molecular Masses ◽  
Cold Inactivation

The thermal, kinetic, and electrophoretic properties of phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) were analyzed in plants from two ecotypes of barnyard grass (Echinochloa crus-galli (L.) Beauv.: Poaceae) originated from sites of contrasting climates in Quebec (QUE) and Mississippi (MISS). The thermostability, cold inactivation, the apparent energy of activation (Ea), the Michaelis-Menten constant (Km), and Vmax/Kmratios for phosphoenolpyruvate (PEP) and Mg2+were analyzed with desalted Sephadex G-25 crude PEPC extracts, with partially purified PEPC from the polyethylene glycol (PEG) 13% fraction and with purified PEPC obtained after elution from DEAE-Sepharose affinity chromatography. PEPC from illuminated leaves from both ecotypes consisted of one isomorph with the same electrophoretic mobility in polyacrylamide gels, similar molecular masses for the native enzyme (400 kDa) and for each subunit of the tetramer (100 kDa), and a same isoelectric point (pI) of 4.95. The only kinetic property for PEPC for which differences of physiological importance among ecotypes were observed at the three levels of purification was Kmfor PEP for which values for QUE plants were significantly lower at low assay temperatures. Differences among ecotypes for thermostability were only observed in assays with crude and partially purified PEPC extracts, while no differences were found for cold inactivation rates, Km(Mg2+) estimates at any level of purification or for Vmax/Kmratios (PEP or Mg2+) from purified PEPC. Significant differences among the two ecotypes were found for catalytic constant (Kcat) estimates obtained with purified PEPC. However, results show higher catalytic efficiency for PEPC from MISS plants at high assay temperatures but no indication of an improved catalytic efficiency for PEPC from QUE plants at low assay temperatures. The lack of ecotypic differences for most thermal and kinetic properties observed with purified PEPC casts doubts about the evolutionary interpretations of results obtained in previous kinetic comparative analyses, which were based on crude or partially purified enzymatic preparations of PEPC extracted from E. crus-galli plants.Key words: phosphoenolpyruvate carboxylase, enzyme kinetics, thermal adaptation, barnyard grass, electrophoresis, Echinochloa crus-galli.

Molecular Forms and Kinetic Properties of Pyruvate, PI Dikinase From Two Populations of Barnyard Grass (Echinochloa crus-galli) From Sites of Contrasting Climates

1996 ◽  
Vol 23 (2) ◽  
pp. 191 ◽  
Author(s):  
JP Simon
Keyword(s):  
Specific Activity ◽  
Molecular Forms ◽  
Kinetic Properties ◽  
Activity Levels ◽  
Light Activation ◽  
Turnover Number ◽  
Barnyard Grass ◽  
Molecular Weights ◽  
Kinetics Of ◽  
Two Populations

Plants from two populations of the C4 barnyard grass (Echinochloa crus-galli (L.) Beauv.) from Qu�bec (QUE) and Mississippi (MISS) were acclimated under controlled conditions to 26/20 and 14/8�C daylnight. The apparent energy of activation (Ea, Km for pyruvate, Vmax/Km ratios, Kcat (substrate turnover number) and specific activity of pyruvate, PI dikinase (PPDK, EC 2.7.9.1) were analysed from partially purified Sephadex G-25 extracts of PPDK from leaves and from highly purified PPDK. PPDK from both populations consisted of one isomorph with the same electrophoretic mobility in polyacrylamide gels and similar molecular weights for the native enzyme (385 kDa) and for the subunit of the tetramer (94.8 kDa). No significant differences were observed for any of the kinetic properties of partially purified or purified PPDK or for the specific activity per mg protein of purified PPDK extracted from plants of the two populations and acclimated to the two thermoperiods. Net photosynthetic rates (Ps) were positively correlated with PPDK activity levels (E) but ElPs ratios were lower than 1.0, ranging from 0.43 to 0.67. Results indicate that differences in activity levels, thermal properties and in the kinetics of light activation and dark inactivation of PPDK extracted from cold-acclimated MISS and QUE plants, as reported in earlier studies, are due to causes other than kinetic properties or electrophoretic characteristics of PPDK.

Temperature Effects on the Activation and Inactivation of Pyruvate, Pi Dikinase in Two Populations of the C4 Weed Echinochloa crus-galli (Barnyard Grass) From Sites of Contrasting Climates

1994 ◽  
Vol 21 (4) ◽  
pp. 463 ◽  
Author(s):  
JP Simon ◽  
MD Hatch
Keyword(s):  
Temperature Effects ◽  
C4 Photosynthesis ◽  
Cold Sensitivity ◽  
Significant Finding ◽  
Initial Activity ◽  
Barnyard Grass ◽  
Two Populations ◽  
Cold Inactivation ◽  
Rate Of Activation

Five-week-old plants of Echinochloa crus-galli (L.) Beauv. (barnyard grass) from Mississippi (MISS) and from Québec (QUE) grown under controlled conditions were subjected to cold temperature acclimatory treatments for periods of up to 3 days. After plants were transferred from a 26/20�C day/night regime to 14/8�C for 2-3 days the rate of activation of the C4 photosynthetic enzyme pyruvate, Pi dikinase (PPDK; EC 2.7.9.1), following illumination of plants at 26�C, was substantially reduced in both ecotypes. This effect was far more pronounced in MISS plants and the PPDK activity of these plants remained at less than half that of control plants (26/20�C) even after 2 h illumination. After 3 days at 14/8�C the half-time for PPDK activation was more than 120 min in MISS plants and 35 min in QUE plants compared with about 3 min for plants remaining in the 26/20�C regime. Lesser but qualitatively similar effects were observed when plants were exposed to only cooler nights (26/8�C) or cooler days (14/20�C). When plants at the 14/8�C regime were transferred back to 26/20�C for 2 days there was a substantial recovery of the capacity for rapid PPDK activation but recovery was slower in MISS plants. Predictably, the rate of activation of PPDK was reduced when activation in control plants (26/20�C) was carried out at 8�C instead of 26�C. However, a significant finding was that the rate of activation at 8�C was more strongly affected in MISS plants, with a reduction to about 15% of the rate of control plants and a final steady PPDK level after 60 min of only half that in control plants. PPDK extracted from MISS plants underwent more rapid in vitro cold inactivation (2�C) than the enzyme from QUE plants and only 80% of the initial activity was recovered upon rewarming at 25�C. The physiological significance of these results are discussed in relation to the cold sensitivity of C4 photosynthesis and the previously reported differences in the growth and physiological performance of MISS and QUE plants at lower temperatures.

Kinetic characterization and regulation of the human retinaldehyde dehydrogenase 2 enzyme during production of retinoic acid

2016 ◽  
Vol 473 (10) ◽  
pp. 1423-1431 ◽  
Author(s):  
Yehuda Shabtai ◽  
Halim Jubran ◽  
Taher Nassar ◽  
Joseph Hirschberg ◽  
Abraham Fainsod
Keyword(s):  
Retinoic Acid ◽  
Developmental Disorders ◽  
Catalytic Efficiency ◽  
Early Embryo ◽  
Activity Level ◽  
In Vitro Assays ◽  
Kinetic Properties ◽  
Retinaldehyde Dehydrogenase

Retinoic acid (RA) is an important regulator of embryogenesis and tissue homoeostasis. Perturbation of RA signalling causes developmental disorders, osteoarthritis, schizophrenia and several types of tumours. RA is produced by oxidation of retinaldehyde from vitamin A. The main enzyme producing RA in the early embryo is retinaldehyde dehydrogenase 2 (RALDH2, ALDH1A2). In the present study we describe in depth the kinetic properties and regulation of the human RALDH2 (hRALDH2) enzyme. We show that this enzyme produces RA using in vivo and in vitro assays. We studied the naturally occurring all-trans-, 9-cis- and 13-cis-retinaldehyde isomers as substrates of hRALDH2. Based on the values measured for the Michaelis–Menten constant Km and the maximal rate Vmax, in vitro hRALDH2 displays the same catalytic efficiency for their oxidation. We characterized two known inhibitors of the vertebrate RALDH2 and determined their kinetic parameters on hRALDH2. In addition, RA was studied as a possible inhibitor of hRALDH2 and a regulator of its activity. We show that hRALDH2 is not inhibited by its oxidation product, all-trans-RA, suggesting the absence of a negative feedback regulatory loop. Expression of the Raldh2 gene is known to be regulated by RA itself, suggesting that the main regulation of the hRALDH2 activity level is transcriptional.

In Vitro Diabetogenic Effect of Cadmium on Liver

2017 ◽  
Vol 8 (01) ◽  
Author(s):  
Agung Biworo ◽  
Dwi Rezki Amalia ◽  
Gratianus Billy Himawan ◽  
Lisda Rizky Amalia ◽  
Valentina Halim ◽  
...  
Keyword(s):  
Glucose Metabolism ◽  
Liver Homogenate ◽  
Liver Cells ◽  
Male Rats ◽  
Glycogen Level ◽  
Liver Sample ◽  
Km Value ◽  
Cd Exposure ◽  
Menten Constant

The objectives of this study were to determine the effect of cadmium (Cd) on glucose metabolism disruption in liver cells homogenate in vitro. The glucose metabolism disruption was analyzed by measuring the level of liver glucose, glycogen and methylglyoxal (MG), and the activity of glucokinase activity. In this experiment, a liver sample was taken from male rats (Rattus novergicus). Samples then homogenized and divided into four groups with; C served as control which contains liver homogenate only; T1 which contains liver homogenate + 0.03 mg/l of cadmium sulphate (CdSO4); T2 which contains liver homogenate + 0.3 mg/l of CdSO4; and T3 which contains liver homogenate + 3 mg/l of CdSO4. After treatment, liver glucose, glycogen, and MG levels, and glucokinase activity were estimated. The activity of liver glucokinase was estimated by measuring the Michaelis-Menten constant (Km) value. The results revealed that Cd exposure could significantly increase glucose and MG levels, the Km value of glucokinase, and decreased the glycogen level in liver cells (P>0.05). These results indicated that Cd exposure induced the disruption of glucose metabolism in the liver.

scholarly journals Engineering subtilisin proteases that specifically degrade active RAS

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Yingwei Chen ◽  
Eric A. Toth ◽  
Biao Ruan ◽  
Eun Jung Choi ◽  
Richard Simmerman ◽  
...  
Keyword(s):  
Active Form ◽  
High Specificity ◽  
Kinetic Properties ◽  
Target Sequence ◽  
Reporter System ◽  
Human Cell Culture ◽  
Conserved Sequence ◽  
Cofactor Binding ◽  
Selective Proteolysis

AbstractWe describe the design, kinetic properties, and structures of engineered subtilisin proteases that degrade the active form of RAS by cleaving a conserved sequence in switch 2. RAS is a signaling protein that, when mutated, drives a third of human cancers. To generate high specificity for the RAS target sequence, the active site was modified to be dependent on a cofactor (imidazole or nitrite) and protease sub-sites were engineered to create a linkage between substrate and cofactor binding. Selective proteolysis of active RAS arises from a 2-step process wherein sub-site interactions promote productive binding of the cofactor, enabling cleavage. Proteases engineered in this way specifically cleave active RAS in vitro, deplete the level of RAS in a bacterial reporter system, and also degrade RAS in human cell culture. Although these proteases target active RAS, the underlying design principles are fundamental and will be adaptable to many target proteins.

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