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.

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.

scholarly journals Carbonic anhydrase isoenzymes in the erthrocytes and dorsolateral prostate of the rat

1969 ◽  
Vol 114 (3) ◽  
pp. 463-476 ◽  
Author(s):  
J. E. A. McIntosh
Keyword(s):  
Carbonic Anhydrase ◽  
High Activity ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Exchange Chromatography ◽  
Kinetic Properties ◽  
Efficient Catalyst ◽  
Molecular Weights ◽  
Hydrolysis Of ◽  
Low Activity

1. Three forms of the zinc-containing enzyme carbonic anhydrase (EC 4.2.1.1) were isolated from the erythrocytes of the rat and two forms from the dorsolateral prostate of the rat. Several additional minor components were observed but not isolated. Separation of the isoenzymes was achieved by ion-exchange chromatography, polyacrylamide-gel electrophoresis and isoelectric focusing. 2. The general properties of the isolated isoenzymes, their molecular weights and their contents of zinc were closely similar. As catalysts of the hydration of carbon dioxide, however, they were distinctly different. The two most abundant isoenzymes of the erythrocytes, which were found in equal proportions, differed 70-fold in specific activity, whereas the isoenzymes of the dorsolateral prostate were similar to one another and resembled the high-activity component of the erythrocytes. The inhibition of the latter by acetazolamide (5-acetamido-1-thia-3,4-diazole-2-sulphonamide) was mainly competitive, whereas in identical conditions the low-activity erythrocyte component and the dorsolateral prostate isoenzymes were non-competitively inhibited. 3. The use of chloroform–ethanol to remove haemoglobin from the rat haemolysate was found (a) to bring about changes in the kinetic properties of the soluble isoenzymes and (b) to cause the appearance of an additional isoenzyme. 4. The actions were compared of the inhibitors acetazolamide, 1,1-dimethylaminonaphthalene-5-sulphonamide and ethoxzolamide (6-ethoxybenzothiazole-2-sulphonamide) on the hydrolysis of p-nitrophenyl acetate catalysed by the isoenzymes. 5. The low-activity erythrocyte isoenzyme was an efficient catalyst of the hydrolysis of β-naphthyl acetate whereas the high-activity forms were much less active towards this ester. Neither of the isoenzymes present in the dorsolateral prostate catalysed this reaction. 6. Carbonic anhydrase in the rat dorsolateral prostate accounts for no more than 5% of the unusually high content of zinc in this organ.

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.

scholarly journals Kinetics of carbonyl reductase from human brain

1987 ◽  
Vol 244 (1) ◽  
pp. 165-171 ◽  
Author(s):  
K M Bohren ◽  
J P von Wartburg ◽  
B Wermuth
Keyword(s):  
Isotope Effects ◽  
Product Inhibition ◽  
Carbonyl Reductase ◽  
Molecular Forms ◽  
Kinetic Properties ◽  
Rate Analysis ◽  
Sequential Mechanism ◽  
Straight Lines ◽  
Inhibition Studies ◽  
Kinetics Of

Initial-rate analysis of the carbonyl reductase-catalysed reduction of menadione by NADPH gave families of straight lines in double-reciprocal plots consistent with a sequential mechanism being obeyed. The fluorescence of NADPH was increased up to 7-fold with a concomitant shift of the emission maximum towards lower wavelength in the presence of carbonyl reductase, and both NADPH and NADP+ caused quenching of the enzyme fluorescence, indicating formation of a binary enzyme-coenzyme complex. Deuterium isotope effects on the apparent V/Km values decreased with increasing concentrations of menadione but were independent of the NADPH concentration. The results, together with data from product inhibition studies, are consistent with carbonyl reductase obeying a compulsory-order mechanism, NADPH binding first and NADP+ leaving last. No significant differences in the kinetic properties of three molecular forms of carbonyl reductase were detectable.

Soluble Thrombomodulin Purified from Human Urine Exhibits a Potent Anticoagulant Effect In Vitro and In Vivo

1995 ◽  
Vol 73 (05) ◽  
pp. 805-811 ◽  
Author(s):  
Yasuo Takahashi ◽  
Yoshitaka Hosaka ◽  
Hiromi Niina ◽  
Katsuaki Nagasawa ◽  
Masaaki Naotsuka ◽  
...  
Keyword(s):  
Human Urine ◽  
Specific Activity ◽  
Anticoagulant Activity ◽  
Rabbit Lung ◽  
Soluble Thrombomodulin ◽  
Molecular Weights ◽  
Dose Dependent Fashion ◽  
Dose Dependent

SummaryWe examined the anticoagulant activity of two major molecules of soluble thrombomodulin purified from human urine. The apparent molecular weights of these urinary thrombomodulins (UTMs) were 72,000 and 79,000, respectively. Both UTMs showed more potent cofactor activity for protein C activation [specific activity >5,000 thrombomodulin units (TMU)/mg] than human placental thrombomodulin (2,180 TMU/mg) and rabbit lung thrombomodulin (1,980 TMU/mg). The UTMs prolonged thrombin-induced fibrinogen clotting time (>1 TMU/ml), APTT (>5 TMU/ml), TT (>5 TMU/ml) and PT (>40 TMU/ml) in a dose-dependent fashion. These effects appeared in the concentration range of soluble thrombomodulins present in human plasma and urine. In the rat DIC model induced by thromboplastin, administration of UTMs by infusion (300-3,000 TMU/kg) restored the hematological abnormalities derived from DIC in a dose-dependent fashion. These results demonstrate that UTMs exhibit potent anticoagulant and antithrombotic activities, and could play a physiologically important role in microcirculation.

Low Molecular Weight Trypsin-Plasmin Inhibitors Isolated from Papain Treated Urinary Trypsin Inhibitor

1982 ◽  
Vol 47 (01) ◽  
pp. 014-018 ◽  
Author(s):  
H Sumi ◽  
N Toki ◽  
S Takasugi ◽  
S Maehara ◽  
M Maruyama ◽  
...  
Keyword(s):  
Trypsin Inhibitor ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Chromatography ◽  
Urinary Trypsin Inhibitor ◽  
Plasmin Activity ◽  
Molecular Weights ◽  
Plasmin Inhibitors

SummaryPapain treatment of human urinary trypsin inhibitor (UTI67; mol. wt. 43,000 by SDS-polyacrylamide gel electrophoresis, specific activity 1,897 U/mg protein) produced four new protease inhibitors, which were highly purified by gel chromatography on Sephadex G-100 and isoelectric focusing. The purified inhibitors (UTI26, UTI9-I, UTI9-II, and UTI9-III) were shown to be homogeneous by polyacrylamide disc gel electrophoresis, and had apparent molecular weights of 26,000, 9,000, 9,000, and 9,800, respectively, by sodium dodecyl sulfate gel electrophoresis. During enzymatic degradation of UTI67, the amino acid compositions changed to more basic, and the isoelectric point increased from pH 2.0 (UTI67) to pHs 4.4, 5.2, 6.6, and 8.3 (UTI26, UTI9-I, UTI9-II, and UTI9-III), respectively. Both the parent and degraded inhibitors had anti-plasmin activity as well as antitrypsin and anti-chymotrypsin activities. Much higher anti-plasmin/anti-trypsin and anti-plasmin/anti-chymotrypsin activities were observed in the degraded inhibitors than in the parent UTI67. They competitively inhibited human plasmin with Ki values of 1.13 X 10-7 - 2.12 X 10-6 M (H-D-Val-Leu-Lys-pNA substrate). The reactions were very fast and the active site of the inhibitors to plasmin was thought to be different from that to trypsin or chymotrypsin.

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