Isolation of plant lactate dehydrogenase by affinity chromatography and role of histidine in the molecule of the enzyme

1980 ◽  
Vol 45 (5) ◽  
pp. 1608-1615 ◽  
Author(s):  
Jana Barthová ◽  
Pavla Plachá ◽  
Sylva Leblová
Keyword(s):  
Lactate Dehydrogenase ◽  
Affinity Chromatography ◽  
Inactivation Rate ◽  
Molar Ratio ◽  
Coenzyme Binding ◽  
Soybean Seedlings ◽  
Diethyl Pyrocarbonate ◽  
Binary Complexes ◽  
Sepharose 4B

Lactate dehydrogenase (EC 1.1.1.27) was isolated from soybean seedlings (Glycine max. L.) by affinity chromatography on an AMP-Sepharose 4B column. The enzyme obtained was inactivated by treatment with diethyl pyrocarbonate; the inactivation rate was proportional to the molar ratio of the enzyme to the reagent. The plot of the inactivation rate versus pH shows that of all the functional groups of the protein the imidazole groups of histidine only were modified by diethyl pyrocarbonate. By this procedure 20 histidine residues were ethoxyformylated in the molecule of soybean lactate dehydrogenase yet 8 only, i.e. two in every subunit were essential for thae activity of the enzyme. A comparison of the effect of diethyl pyrocarbonate on the lactate dehydrogenase apoenzyme with its effect on the binary complexes of the enzyme with coenzymes or on ternary complexes with its both substrates permits the conclusion that histidine is involved not only in the proton transfer during the redox reaction but also in the coenzyme-binding site.

Improved purification of human lactate dehydrogenase isoenzyme-3 and studies with its fluorescein isothiocyanate and biotin conjugates

1990 ◽  
Vol 36 (1) ◽  
pp. 59-64
Author(s):  
R N Weijers ◽  
R de Bruijn ◽  
J Mulder ◽  
H Kruijswijk
Keyword(s):  
Amino Acid ◽  
Lactate Dehydrogenase ◽  
Affinity Chromatography ◽  
B Lymphocytes ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Fluorescein Isothiocyanate ◽  
Molar Ratio ◽  
The Stability ◽  
Sepharose 4B

Abstract Lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) isoenzyme-3 (LD-3) has been isolated in milligram quantities from human erythrocytes. Using an improved procedure--which involves complete hemolysis of the erythrocytes, diethylaminoethyl (DEAE)-Sephacel column chromatography, and 5'-AMP-Sepharose 4B affinity chromatography--we obtained 23,000-fold purified isoenzyme from the crude hemolysate (overall yield about 90%). The final product was homogeneous on polyacrylamide disc gel electrophoresis and had a specific activity of about 435 kU/g. Its amino acid composition is presented. With the eventual aim to make visible and isolate IgA kappa antibody-secreting B lymphocytes, we developed reproducible methods for preparing fluorescein isothiocyanate isomer-1-conjugated LD-3 with a fluorescein/LD-3 molar ratio between 1.3 and 3.3, and biotinylated LD-3 with a biotin/LD-3 molar ratio between 1.3 and 2.5. In evaluating the stability of these two conjugates, we determined that they still can react with IgA kappa to form the IgA kappa (LD-3)2 complex.

Purification Of Vasoactive Peptides From Human Fibrinogen Degraded By Human Leucocyte Elastase

1981 ◽  
Author(s):  
R Wallin ◽  
M Belew ◽  
K Ohlsson ◽  
T Saldeen
Keyword(s):  
Affinity Chromatography ◽  
Vascular Permeability ◽  
Gel Filtration ◽  
Molar Ratio ◽  
Significant Activity ◽  
Small Peptides ◽  
Human Fibrinogen ◽  
Leucocyte Elastase ◽  
Pm 10 ◽  
Sepharose 4B

The presence of leucocytes around extravascular fibrin deposits suggests that the leucocyte elastases might be partly responsible for the extravascular degradation of fibrin. Our previous studies have shown that the degradation of fibrin(ogen) by plasmin leads to the release of 2 small peptides which markedly increase vascular permeability and induce oedema e.g. in the lungs. The results of this investigation show that small peptides released from fibrinogen after degradation by leucocytes elastases also increase vascular permeability.Human fibrinogen (Kabi, Grade L) was made plasminogenfree by affinity chromatography on Lysine-Sepharose 4B prior to use. The human leucocyte elastases were isolated from extracts of lysosome-like granules of human leukaemic myeloid cells by a combination of gel filtration, affinity chromatography and preparative agarose gel electrophoresis. The fibrinogen (0.5 %) and the leucocyte elastases (in a molar ratio of 100:1) were incubated together for 48 h at +37°C and at pH 8.5. The mixture was then cooled to +4°C to stop the lysis and ultrafiltrated on a DIAFLO PM 10 membrane until the retentate was approximately 10 % of the starting volume. The peptides in the diffusate accounted for about 20 % of the starting material as estimated from absorbance measurements at 280 nm. The diffusate was concentrated by lyophilization and fractionated by chromatography on a column of Bio-Gel P-6. At least 8 fractions were obtained of which only two showed a significant activity in their ability to increase vascular permeability in rat skin. The active peptides in these two fractions were further purified to homogeneity by column zone electrophoresis at various pHs and their amino acid compositions established.

A lactate dehydrogenase-immunoglobulin G1 complex, not blocked by anti-idiotype antibody, in a patient with IgG1-lambda type M-proteinemia.

1987 ◽  
Vol 33 (8) ◽  
pp. 1478-1483 ◽  
Author(s):  
K Fujita ◽  
I Sakurabayashi ◽  
M Kusanagi ◽  
T Kawai
Keyword(s):  
Lactate Dehydrogenase ◽  
Complex Formation ◽  
Affinity Chromatography ◽  
Binding Site ◽  
Binding Capacity ◽  
Isoenzyme Pattern ◽  
M Protein ◽  
Light Chains ◽  
Ldh Isoenzymes ◽  
Sepharose 4B

Abstract The serum of a patient with IgG1-lambda type M-proteinemia showed an abnormal isoenzyme pattern for lactate dehydrogenase (LDH, EC 1.1.1.27). By affinity chromatography, we showed that four isoenzymes (LDH2, LDH3, LDH4, and LDH5) were bound to the M-protein. This complex formation was not blocked by anti-idiotype antibody, even though the binding capacity of IgG was exclusively located in the Fab region of the molecule. Moreover, heavy and light chains of the patient's IgG, obtained by reduction, separately had affinities for each of the LDH isoenzymes. LDH-IgG complex was easily dissociated by affinity chromatography on 5'-AMP-Sepharose 4B or by added NADH. We propose the following hypothesis for the LDH-IgG complex formation: LDH can recognize the gamma-Fab region of IgG at the NAD+ binding site of the molecule, but the affinity of the LDH molecule for immunoglobulin is much weaker than that for NADH or 5'-AMP.

Role of histidine in molecule of pea alcohol dehydrogenase

1982 ◽  
Vol 47 (5) ◽  
pp. 1408-1413 ◽  
Author(s):  
Noemi Čeřovská ◽  
Jana Barthová ◽  
Sylva Leblová
Keyword(s):  
Alcohol Dehydrogenase ◽  
Enzymatic Activity ◽  
Inactivation Rate ◽  
Histidine Residue ◽  
Michaelis Constant ◽  
Diethyl Pyrocarbonate ◽  
Inactivation Rate Constant ◽  
Pea Seedlings ◽  
Absorbance Difference

Alcohol dehydrogenase (E.C.1.1.1.1) from germinating pea seedlings was modified by treatment with diethyl pyrocarbonate. The inactivation rate is proportional to the molar concentration of the modifying agent; the inactivation was almost complete in fifty minutes at a diethyl pyrocarbonate concentration of 5 . 10-6 mol/l. The histidine content calculated from the absorbance difference at 240 nm was 3.43 residues per molecule of native and 4.75 residues per molecule of demetalized enzyme. A correlation of the absorbance difference at 240 nm with a 100% loss of enzymatic activity shows that 1.22 histidine residue is essential for the activity of alcohol dehydrogenase. The dependence of the inactivation rate constant on the pH of the medium indicates that the treatment of pea alcohol dehydrogenase with diethyl pyrocarbonate results in the modification of one group only with a pK of 7.1, well corresponding to the imidazole group of histidine. The enzyme is partially protected against inactivation by NADH at a concentration close to the Michaelis constant for NADH. The treatment of the ethoxyformylated enzyme with hydroxylamine followed by dialysis restored the activity of pea alcohol dehydrogenase by 88%.

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.

The use of Spheron as a matrix for affinity chromatography of NAD-dependent dehydrogenases

1986 ◽  
Vol 51 (7) ◽  
pp. 1542-1549
Author(s):  
Jan Kovář ◽  
Alena Škodová ◽  
Jaroslav Turánek
Keyword(s):  
Lactate Dehydrogenase ◽  
Alcohol Dehydrogenase ◽  
Affinity Chromatography ◽  
Dehydrogenase Activity ◽  
Nitrous Acid ◽  
Separation Efficiency ◽  
Binding Capacity ◽  
The Stability ◽  
Sepharose 4B

The paper compares several methods of coupling common ligands of dehydrogenases, viz. N6-[(6-aminohexyl)carbamoylmethyl]-AMP and N6-[(6-aminohexyl)carbamoylmethyl]-NAD, to a hydrophilic macroporous glycolmethacrylate gel, Spheron. The affinants coupled best to a CNBr-activated gel and to a gel with hydrazine groups (after activation with nitrous acid). The affinity properties of gels based on Spheron and on Sepharose 4B were similar ( the stability and separation efficiency were almost identical, the binding capacity and the recovery of dehydrogenase activity were somewhat better with the Sepharose). The materials based on Spheron were used in several separation experiments, viz. separation of lactate dehydrogenase form albumin, separation of lactate dehydrogenase from alcohol dehydrogenase under different conditions and separation of isoenzymes of lactate dehydrogenase. Spheron 300 with a coupled affinant was also employed in an attempt to purify a crude alcohol dehydrogenase.

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.

Quantitative Assessment of Soluble Fibrin in Plasma by Affinity Chromatography – A Comparative Study with desAA-Fibrin, desAABB-Fibrin and Fibrinogen

1985 ◽  
Vol 54 (02) ◽  
pp. 533-538 ◽  
Author(s):  
Wilfried Thiel ◽  
Ulrich Delvos ◽  
Gert Müller-Berghaus
Keyword(s):  
Affinity Chromatography ◽  
Calcium Ions ◽  
Quantitative Assessment ◽  
Fluid Phase ◽  
Soluble Fibrin ◽  
Binding Characteristics ◽  
Different Temperatures ◽  
Immobilized Proteins ◽  
Sepharose 4B

SummaryA quantitative determination of soluble fibrin in plasma was carried out by affinity chromatography. For this purpose, desAA-fibrin and fibrinogen immobilized on Sepharose 4B were used at the stationary side whereas batroxobin-induced 125I-desAA-fibrin or thrombin-induced 125I-desAABB-fibrin mixed with plasma containing 131I-fibrinogen represented the fluid phase. The binding characteristics of these mixtures to the immobilized proteins were compared at 20° C and 37° C. Complete binding of both types of fibrin to the immobilized desAA-fibrin was always seen at 20° C as well as at 37° C. However, binding of soluble fibrin was accompanied by substantial binding of fibrinogen that was more pronounced at 20° C. Striking differences depending on the temperature at which the affinity chromatography was carried out, were documented for the fibrinogen-fibrin interaction. At 20° C more than 90% of the applied desAA-fibrin was bound to the immobilized fibrinogen whereas at 37° C only a mean of 17% were retained at the fibrinogen-Sepharose column. An opposite finding with regard to the tested temperature was made with the desAABB-fibrin. Nearly complete binding to insolubilized fibrinogen was found at 37° C (95%) but only 58% of the desAABB-fibrin were bound at 20° C. The binding patterns did not change when the experiments were performed in the presence of calcium ions. The opposite behaviour of the two types of soluble fibrin to immobilized fibrinogen at the different temperatures, together with the substantial binding of fibrinogen in the presence of soluble fibrin to insolubilized fibrin in every setting tested, devaluates affinity chromatography as a tool in the quantitative assessment of soluble fibrin in patients’ plasma.

Role of reduced precursor and solvolytic reagent molar ratio on preparation and properties of ionogel

2016 ◽  
Vol 242 ◽  
pp. 29-37 ◽  
Author(s):  
Abhishek Kumar Gupta ◽  
Yogendra Lal Verma ◽  
Manish Pratap Singh ◽  
Rajendra Kumar Singh
Keyword(s):  
Molar Ratio

scholarly journals Evaluation of equilibrium constants for the interaction of lactate dehydrogenase isoenzymes with reduced nicotinamide-adenine dinucleotide by affinity chromatography

1975 ◽  
Vol 151 (3) ◽  
pp. 631-636 ◽  
Author(s):  
R I Brinkworth ◽  
C J Masters ◽  
D J Winzor
Keyword(s):  
Lactate Dehydrogenase ◽  
Affinity Chromatography ◽  
Equilibrium Constants ◽  
Mouse Tissue ◽  
Rabbit Muscle ◽  
Muscle Lactate ◽  
Sodium Phosphate Buffer ◽  
A Value ◽  
Series Of Experiments ◽  
Reduced Nicotinamide Adenine Dinucleotide

Rabbit muscle lactate dehydrogenase was subjected to frontal affinity chromatography on Sepharose-oxamate in the presence of various concentrations of NADH and sodium phosphate buffer (0.05 M, pH 6.8) containing 0.5 M-NaCl. Quantitative interpretation of the results yields an intrinsic association constant of 9.0 × 104M−1 for the interaction of enzyme with NADH at 5°C, a value that is confirmed by equilibrium-binding measurements. In a second series of experiments, zonal affinity chromatography of a mouse tissue extract under the same conditions was used to evaluate assoication constants of the order 2 × 105M−1, 3 × 105M−1, 4 × 105M−1, 7 × 105M−1 and 2 × 106M−1 for the interaction of NADH with the M4, M3H, M2H2, MH3 and H4 isoenzymes respectively of lactate dehydrogenase.

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