Interaction of lactate dehydrogenase isoenzymes with ligands

1988 ◽  
Vol 53 (8) ◽  
pp. 1857-1861 ◽  
Author(s):  
Jana Barthová ◽  
Jana Kučerová ◽  
Sylva Leblová
Keyword(s):  
Lactate Dehydrogenase ◽  
Enzyme Inhibitor ◽  
Dissociation Constants ◽  
Physiological Role ◽  
Competitive Inhibitor ◽  
Kinetic Measurements ◽  
Cibacron Blue ◽  
Michaelis Constants ◽  
Sepharose 4B ◽  
Lactate Dehydrogenase Isoenzymes

Isoenzymes of bovine lactate dehydrogenase (H4, H3M, and H2M2) were prepared by affinity chromatography on a 5'-AMP-Sepharose 4B column. The interaction of isoenzymes with two ligands, coenzyme NADH and the competitive inhibitor Cibacron Blue F3GA was followed by means of kinetic measurements and by affinity electrophoresis. The Michaelis constants of NADH were compared with the inhibition constants of Cibacron Blue and dissociation constants of enzyme-inhibitor complexes. It was found that the M subunit of lactate dehydrogenase exhibits always higher affinity both to NADH and Cibacron Blue in comparison to the H subunit. This finding corresponds to the physiological role of lactate dehydrogenase isoenzymes.

Modification of arginine residues of pea lactate dehydrogenase by phenylglyoxal

1990 ◽  
Vol 55 (5) ◽  
pp. 1380-1388
Author(s):  
Jana Barthová ◽  
Jana Kunová ◽  
Sylva Leblová
Keyword(s):  
Lactate Dehydrogenase ◽  
Dissociation Constants ◽  
Ionic Bond ◽  
Complete Inactivation ◽  
Pea Seedlings ◽  
Arginine Residues ◽  
Michaelis Constants ◽  
Lactate And Pyruvate ◽  
Sepharose 4B

Electrophoretically homogeneous lactate dehydrogenase was isolated from germinating pea seedlings by chromatography on AMP-Sepharose 4B. The amino acid composition of the enzyme was determined as well as the values of the Michaelis constants for four substrates and of the dissociation constants for the binary enzyme-coenzyme complexes. The treatment of the enzyme with phenylglyoxal resulted in the modification of nine arginine residues in its subunit. The modification was paralleled by a complete inactivation of the enzyme. The role of the arginine residues in the active center probably involves the binding of substrates, lactate and pyruvate, to the apoenzyme by an ionic bond.

Study of porous cellulose beads as a dye-ligand matrix. Effect of protein admixtures and concentration of immobilized dye in the quantitative analysis of lactate dehydrogenase: Cibacron blue interaction

1990 ◽  
Vol 55 (2) ◽  
pp. 581-586 ◽  
Author(s):  
Danica Mislovičová ◽  
Peter Gemeiner ◽  
Viera Ďurišová
Keyword(s):  
Quantitative Analysis ◽  
Lactate Dehydrogenase ◽  
Matrix Effect ◽  
Dissociation Constants ◽  
Cibacron Blue ◽  
Blue Dextran ◽  
Nonspecific Interaction ◽  
The Matrix ◽  
Bead Cellulose ◽  
Total Interaction

The effect of protein admixtures and concentration of immobilized dye on the interaction of lactate dehydrogenase (LDH) with Cibacron Blue-bead cellulose has been studied by means of zonal chromatography with Cibacron Blue-dextran T 10 as the mobile ligand. The influence of both variables on the values of dissociation constants of complexes immobilized dye-LDH (KI-L) and mobile dye-LDH (KM-L) can be summarized as follows: The values of KI-L remained practically constant and were found to be lower than those of KM-L. The differences found in KI-L and KM-L values should help to estimate the contribution of nonspecific interaction of the matrix to the total interaction between dye-ligand and the enzyme.

scholarly journals A Competitive Inhibitor of Tyrosinase

1951 ◽  
Vol 4 (4) ◽  
pp. 554 ◽  
Author(s):  
C Warner
Keyword(s):  
Enzyme Inhibitor ◽  
Dissociation Constants ◽  
Competitive Inhibitor ◽  
Enzyme Preparations ◽  
Enzyme Substrate ◽  
Kinetics Of

The kinetics of the activation of catechol by tyrosinase prepared from the potato and the mushroom, and of its inhibition by sodium m-hydroxybenzoate, have been studied. The enzyme-substrate dissociation constants differed markedly between the two enzyme sources (K. potato = 5.OmM, Kg mushroom = O.28mM), as did also the enzyme-inhibitor dissociation constants (K; potato = 2.5mM, Ki mushroom = O.BmM). For both enzyme preparations sodium m-hydroxybenzoate met the requirements of a competitive inhibitor.

scholarly journals General ligands in affinity chromatography. Cofactor–substrate elution of enzymes bound to the immobilized nucleotides adenosine 5′-monophosphate and nicotinamide–adenine dinucleotide

1972 ◽  
Vol 127 (4) ◽  
pp. 625-631 ◽  
Author(s):  
K. Mosbach ◽  
H. Guilford ◽  
R. Ohlsson ◽  
M. Scott
Keyword(s):  
Lactate Dehydrogenase ◽  
Affinity Chromatography ◽  
Cyanogen Bromide ◽  
Competitive Inhibitor ◽  
Single Step ◽  
Lactate Dehydrogenase Activity ◽  
Step Procedure ◽  
The Matrix ◽  
Subsequent Elution ◽  
Sepharose 4B

1. Two different gels have been prepared suitable for the separation of a number of enzymes, in particular NAD+-dependent dehydrogenases, by affinity chromatography. For both the matrix used was Sepharose 4B. For preparation (a), NAD+–Sepharose, 6-aminohexanoic acid has been coupled to the gel by the cyanogen bromide method and then NAD+ was attached by using dicyclohexylcarbodi-imide; for preparation (b), AMP–Sepharose, N6-(6-aminohexyl)-AMP has been coupled directly to cyanogen bromide-activated gel. 2. Affinity columns of both gels retain only the two enzymes when a mixture of bovine serum albumin, lactate dehydrogenase and glyceraldehyde 3-phosphate dehydrogenase is applied. Subsequent elution with the cofactor NAD+ yields glyceraldehyde 3-phosphate dehydrogenase whereas lactate dehydrogenase is eluted by applying the same molarity of the reduced cofactor. 3. The binding of both glyceraldehyde 3-phosphate dehydrogenase and lactate dehydrogenase to the gel tested, AMP–Sepharose, is strong enough to resist elution by gradients of KCl of up to at least 0.5m. A 0.0–0.15m gradient of the competitive inhibitor salicylate, however, elutes both enzymes efficiently and separately. 4. The elution efficiency of lactate dehydrogenase from AMP–Sepharose has been examined by using a series of eluents under comparable conditions of concentration etc. The approximate relative efficiencies are: 0 (lactate); 0 (lactate+semicarbazide); 0 (0.5mm-NAD+); 80 (lactate+NAD+); 95 (lactate+semicarbazide+NAD+); 100 (0.5mm-NADH). 5. All contaminating lactate dehydrogenase activity can be removed from commercially available crude pyruvate kinase in a single-step procedure by using AMP–Sepharose.

scholarly journals Adsorptive pinocytosis of 125I-labelled lactate dehydrogenase isoenzymes H4 and M4 by rat yolk sacs incubated in vitro

1981 ◽  
Vol 198 (3) ◽  
pp. 587-593 ◽  
Author(s):  
T Kooistra ◽  
K E Williams
Keyword(s):  
Lactate Dehydrogenase ◽  
Hydrophobic Interactions ◽  
Dissociation Constants ◽  
Yolk Sac ◽  
Sinusoidal Cells ◽  
Membrane Complex ◽  
Liver Sinusoidal Cells ◽  
Lactate Dehydrogenase Isoenzymes

The pinocytic uptake of 125I-labelled porcine lactate dehydrogenase isoenzymes H4 and M4 by 17.5-day rat visceral yolk sac incubated in vitro was saturable and binding obeyed Michaelis-Menten kinetics. The uptake characteristics of the two isoenzymes were very similar. For the H4 and the M4 isoenzymes, the dissociation constants of the protein-plasma-membrane complex were 0.62 microM and 0.84 microM respectively, and the maximum rates of uptake 0.13 and 0.26 nmol/mg of yolk-sac protein per h respectively. These findings contrast with those from studies in vivo, which show the M4 form is taken up by rat liver sinusoidal cells at a much higher rate than the H4 form, and point to different recognition systems for the adsorptive pinocytosis of simple non-conjugate proteins in yolk-sac epithelial cells and liver sinusoidal cells. Competition experiments indicate that binding of the H4 isoenzyme to the yolk-sac cells is restricted to hydrophobic interactions, whereas the binding of the M4 isoenzyme involves hydrophobic as well as positively charged sites on the protein molecules.

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