scholarly journals Properties of a halophil nicotinamide–adenine dinucleotide phosphate-specific isocitrate dehydrogenase. Preliminary studies of the salt relations and kinetics of the crude enzyme

1970 ◽  
Vol 116 (1) ◽  
pp. 125-134 ◽  
Author(s):  
D. M. Aitken ◽  
A. J. Wicken ◽  
A. D. Brown
Keyword(s):  
Sodium Chloride ◽  
Potassium Chloride ◽  
Isocitrate Dehydrogenase ◽  
Nicotinamide Adenine Dinucleotide ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Competitive Inhibitor ◽  
Michaelis Constants ◽  
The Hill ◽  
Kinetics Of ◽  
Pronounced Inhibition

The effects of chlorides on NADP-specific isocitrate dehydrogenase from Halobacterium salinarium were investigated. The enzyme is stabilized by potassium chloride and sodium chloride and this effect is discussed in relation to the Hill (1913) equation. Kinetics of the enzyme were studied within a range of concentrations of potassium chloride and sodium chloride. Apparent Michaelis constants for both substrates were affected by salt concentration, the effect being greater in sodium chloride than in potassium chloride. Minimal apparent Michaelis constants for both substrates were similar to the corresponding constants reported for yeast isocitrate dehydrogenase. Vmax. was maximal in each salt at a concentration of about 1m. The maximum was higher in sodium chloride than in potassium chloride. At salt concentrations above about 2.3m, the apparent Vmax. was lower in sodium chloride than in potassium chloride, and at salt concentrations below 0.75–1.0m, each salt behaved as a linear activator of the enzyme. Within this concentration range salt and NADP+ acted competitively; the activation by salt was overcome at finite concentrations of NADP+. At concentrations above about 1m, potassium chloride was a linear non-competitive inhibitor of the enzyme. Within the range 1.0–2.5m, sodium chloride was also a linear non-competitive inhibitor, but above 2.5m it caused more pronounced inhibition.

scholarly journals Properties of halophil nicotinamide–adenine dinucleotide phosphate-specific isocitrate dehydrogenase. True Michaelis constants, reaction mechanisms and molecular weights

1972 ◽  
Vol 130 (3) ◽  
pp. 645-662 ◽  
Author(s):  
D. M. Aitken ◽  
A. D. Brown
Keyword(s):  
Reaction Mechanism ◽  
Sodium Chloride ◽  
Potassium Chloride ◽  
Salt Concentration ◽  
Isocitrate Dehydrogenase ◽  
Reaction Mechanisms ◽  
Substrate Concentration ◽  
Gel Filtration ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Michaelis Constants

True values of Michaelis constants of the NADP+-specific isocitrate dehydrogenase from Halobacterium salinarium were not very different from those of the apparent constants reported by Aitken et al. (1970). The true constants were affected by salt in a similar manner to that of the apparent constants obtained with NADP+ at fixed concentrations of 1.0–0.2mm and threo-ds-(+)-isocitrate at fixed concentrations of 2.0–0.125mm. The response of apparent Vmax. to salt concentration was highly dependent on fixed substrate concentration in solutions of sodium chloride but much less so in solutions of potassium chloride. At several levels the results emphasize the difficulty of generalizing about the salt relations of a halophil enzyme without adequate attention to substrate concentration. The enzyme has at least two different reaction mechanisms depending on salt concentration. In its ‘physiological’ form (i.e. in 1.0m-potassium chloride), and also in 1.0m-sodium chloride, the reaction mechanism is ordered with NADP+ the first substrate added and NADPH the last product released. In 0.25m-sodium chloride, however, the mechanism is different and is probably non-sequential. In 4.0m-sodium chloride with low concentrations of either fixed substrate, there was evidence of a co-operative action of the variable substrate. The evidence suggests that salt participates in the reaction mechanism in two ways: one is the reversible addition to the enzyme in a manner analogous to that of a substrate; the other is dead-end complex-formation. The relative contributions of these two types of reaction determine whether salt activates or inhibits the enzyme. In addition, the inhibition caused by high concentrations of sodium chloride is more complex than the corresponding inhibition by potassium chloride. Gel-filtration experiments indicated that at very low salt concentrations the enzyme has an apparent molecular weight of about 70800. In ‘physiological’ concentrations of potassium chloride the enzyme appears to be a dimer (mol.wt. 122000–135000) and, in 1.0–4.0m-sodium chloride, it behaves as a trimer or tetramer (mol.wt. 224000–251000). A preliminary method of purifying the enzyme is described.

scholarly journals Purification and comparative properties of isoenzymes of nicotinamide–adenine dinucleotide phosphate–isocitrate dehydrogenase from rat heart and liver

1972 ◽  
Vol 129 (5) ◽  
pp. 1003-1011 ◽  
Author(s):  
M. Islam ◽  
Joyce L. Bell ◽  
D. N. Baron
Keyword(s):  
Myocardial Infarction ◽  
Ion Exchange ◽  
Ammonium Sulphate ◽  
Isocitrate Dehydrogenase ◽  
Nicotinamide Adenine Dinucleotide ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Differential Sensitivity ◽  
Michaelis Constants ◽  
Ammonium Sulphate Fractionation ◽  
Cytoplasmic Location

1. Rat liver and heart major isoenzymes of NADP–isocitrate dehydrogenase have each been purified about 100-fold by a combination of ammonium sulphate fractionation and chromatography on ion-exchange cellulose and their properties compared. 2. The properties were similar in respect of pH, inhibition by Hg2+and Michaelis constants for isocitrate and NADP. 3. Some of the properties of the isoenzymes were different. 4. The heart isoenzyme was activated about 210% by 0.8m-ammonium sulphate whereas the liver isoenzyme was unaffected. The heart isoenzyme showed greater sensitivity to inactivation by heat (30°C for 30min), whereas the liver isoenzyme was more sensitive to inactivation by p-chloromercuribenzoate and by Cu2+. 5. The Michaelis constants with 3-acetylpyridine–adenine dinucleotide phosphate showed a twofold difference between liver and heart isoenzyme. 6. The differential sensitivity to heat and its mainly non-cytoplasmic location may be an explanation of the failure of plasma isocitrate dehydrogenase activity to increase after a myocardial infarction.

scholarly journals Mitochondrial adenosine triphosphatase from human placenta--inhibition by free magnesium ions of ITP hydrolysis.

1994 ◽  
Vol 41 (1) ◽  
pp. 39-44 ◽  
Author(s):  
Z Aleksandrowicz
Keyword(s):  
Competitive Inhibition ◽  
Competitive Inhibitor ◽  
Hill Coefficient ◽  
Negative Cooperativity ◽  
Magnesium Ions ◽  
Beef Liver ◽  
Bicarbonate Ions ◽  
The Hill ◽  
Kinetics Of ◽  
Free Magnesium

The effects of Mg2+ and bicarbonate on the kinetics of ITP hydrolysis by soluble ATPase (F1) from human placental mitochondria were studied. Increasing amounts of Mg2+ at fixed ITP concentration, caused a marked activation of F1 followed by inhibition at higher Mg2+ concentration. The appropriate substrate for the mitochondrial F1 seems to be the MgITP complex as almost no ITP was hydrolysed in the absence of magnesium. Mg2+ behaved as a competitive inhibitor towards the MgITP complex. In this respect the human placental enzyme differ from that from other sources such as yeast, beef liver or rat liver. The linearity of the plot presenting competitive inhibition by free Mg2+ of MgITP hydrolysis (in the presence of activating bicarbonate anion) suggests that both Mg2+ and MgITP bind to the same catalytic site (Km(MgITP) = 0.46 mM, Ki(Mg) = 4 mM). When bicarbonate was absent in the ITPase assay, placental F1 exhibited apparent negative cooperativity in the presence of 5 mM Mg2+, just as it did with MgATP as a substrate under similar conditions. Bicarbonate ions eliminated the negative cooperativity with respect to ITP (as the Hill coefficient of 0.46 was brought to approx. 1), and thus limited inhibition by free Mg2+. The results presented suggest that the concentration of free magnesium ions may be an important regulatory factor of the human placental F1 activity.

scholarly journals Swelling of polyacrylamide-based hydrogel in aqueous solutions of low-molecular salts

2020 ◽  
Vol 64 (3) ◽  
pp. 293-299
Author(s):  
E. V. Vorobieva
Keyword(s):  
Sodium Chloride ◽  
Potassium Chloride ◽  
Chloride Solution ◽  
Sodium Chloride Solution ◽  
External Solution ◽  
Swelling Kinetics ◽  
Polymer Gel ◽  
Molecular Salts ◽  
Gel Phase ◽  
Kinetics Of

The swelling kinetics of cross-linked polyacrylamide was studied depending on the composition of the external solution. It was shown that the polymer gel swelling during the transition from water to a potassium or sodium chloride solution and vice versa sharply decreases, then gradually increases. The swelling of gel in a solution of sodium chloride is higher than that of potassium chloride. In the equilibrium swollen gel, the concentration of salts in the gel phase is higher than in the external solution and increases with the transition from potassium chloride to sodium chloride.

scholarly journals Purification and properties of the nicotinamide–adenine dinucleotide phosphate-dependent isocitrate dehydrogenase from pig liver cytoplasm

1970 ◽  
Vol 118 (2) ◽  
pp. 253-258 ◽  
Author(s):  
J. A. Illingworth ◽  
K. F. Tipton
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Isocitrate Dehydrogenase ◽  
Nicotinamide Adenine Dinucleotide ◽  
Soluble Fraction ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Turnover Number ◽  
Pig Liver ◽  
Extinction Coefficients ◽  
Purification And Properties

The NADP-dependent isocitrate dehydrogenase from pig liver soluble fraction was purified over 500-fold with an overall yield of 25%. The purified enzyme, which is homogeneous by all the usual criteria, has a molecular weight of about 75000 and is composed of two identical subunits. This has been demonstrated by ultracentrifugation, fluorescence titration and peptide `fingerprinting'. The maximal turnover number, extinction coefficients at 280nm and 260nm and amino acid analysis are described.

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