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.

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.

scholarly journals Clinical and Diagnostic Significance of Lactate Dehydrogenase and Its Isoenzymes in Animals

2020 ◽  
Vol 2020 ◽  
pp. 1-11 ◽  
Author(s):  
Robert Klein ◽  
Oskar Nagy ◽  
Csilla Tóthová ◽  
Frederika Chovanová
Keyword(s):  
Lactate Dehydrogenase ◽  
Cell Injury ◽  
The Body ◽  
Isoenzyme Pattern ◽  
Diagnostic Significance ◽  
Ldh Isoenzymes ◽  
Body Tissues ◽  
Organ Tissue ◽  
Lactate Dehydrogenases

Lactate dehydrogenase (LDH) is widely distributed enzyme in cells of various living systems where it is involved in carbohydrate metabolism catalyzing interconversion of lactate and pyruvate with NAD+/NADH coenzyme system. Cells of tissues are direct source of lactate dehydrogenase isoenzymes that are naturally distributed in blood plasma/serum of animals and humans producing characteristic profile. This profile depends on intracellular isoenzyme concentration in all tissues that contribute to the common pool of lactate dehydrogenases in plasma/serum as a consequence of natural cell degradation. LDH is widely distributed in the body, high activities are found in the heart, liver, skeletal muscle, kidney, and erytrocytes, whereas lesser amounts are found in the lung, smooth muscle, and brain. Because of its widespread activities in numerous body tissues, LDH is elevated in a variety of disorders. There are many conditions that contribute to increased activity of LDH. An elevated total LDH value is a rather nonspecific finding. Therefore, LDH assays assume a more clinical significance when separated into isoenzyme fractions. The activity of LDH and its serum and tissue patterns and composition show great variations between the species. These differences do not allow using catalytic activities of LDH isoenzymes from one species to another. Instead, the pattern of serum LDH isoenzymes should be interpreted in respect to its species origin that is important in particular in veterinary medicine. Determination of total LDH activity and its isoenzyme pattern in serum of mammals had become one of the biochemical indicators in the assessment of organ disorders. When the content of cells is released from tissue to plasma, as on cell injury, the LDH isoenzyme pattern of the serum changes in favour of the profile of the affected organ (tissue) that can be used in the diagnostic practice.

Circulating IgG-LD complex, dissociable by addition of NAD+.

1982 ◽  
Vol 28 (1) ◽  
pp. 236-239 ◽  
Author(s):  
F Gorus ◽  
W Aelbrecht ◽  
B Van Camp
Keyword(s):  
Lactate Dehydrogenase ◽  
Autoimmune Disease ◽  
Complex Formation ◽  
Affinity Chromatography ◽  
Conformational Changes ◽  
Active Site ◽  
Nicotinamide Adenine Dinucleotide ◽  
Binding Sites ◽  
Gel Filtration ◽  
Binding Studies

Abstract Macromolecular LD (lactate dehydrogenase, EC 1.1.1.27) was present in the serum of a patient suffering from idiopathic fibrosis of the lung and presenting signs of autoimmune disease. By using gel filtration and affinity chromatography techniques, the vast majority of the patient's serum LD activity was shown to consist of LD-IgG complexes, which dissociated in the presence of added nicotinamide adenine dinucleotide (NAD+). Binding studies with tritiated NAD+ indicated that complex formation was not ascribable to a lack of circulating cofactor. The most likely explanation for the complex formation was the existence of LD binding sites on IgG molecules. The disruption of the complex by NAD+ might be explained by a competition between IgG molecules and NAD+ for the LD active site or by conformational changes induced in the LD molecules on binding of NAD+.

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.

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 Evaluation of equilibrium constants by affinity chromatography

1974 ◽  
Vol 143 (2) ◽  
pp. 435-443 ◽  
Author(s):  
Lawrence W. Nichol ◽  
Alexander G. Ogston ◽  
Donald J. Winzor ◽  
William H. Sawyer
Keyword(s):  
Affinity Chromatography ◽  
Binding Site ◽  
Ricinus Communis ◽  
Equilibrium Constants ◽  
Quantitative Studies ◽  
Multiple Binding Sites ◽  
Multiple Binding ◽  
Glucose System ◽  
Matrix Bound ◽  
Sepharose 4B

Theoretical expressions are derived for affinity chromatography of systems comprising an acceptor A with one binding site for attachment to a functional group X on the column matrix and one site for interaction with a small ligand B that specifically affects its elution. From a general relationship covering all possible interactions between A, B and X simpler expressions are derived for affinity systems in which only two equilibria operate. Methods are suggested whereby these simpler systems may be characterized in terms of the two pertinent equilibrium constants and the concentration of matrix-bound constituent. The means by which the theory may be adapted to affinity chromatography of acceptors with multiple binding sites for ligand is also illustrated. Results of partition experiments on the Sephadex G-100–lysozyme–d-glucose system in acetate–chloride buffer (I=0.17m), pH5.4, are used to demonstrate the feasibility of evaluating quantitatively affinity-chromatography interactions. Values of 30m−1 and 1.2×106m−1 are obtained for the equilibrium constants for the reactions of lysozyme with glucose and Sephadex respectively, there being only an occasional binding site in the polysaccharide matrix (approximately 1 in 105 glucose residues). In a second experimental study the phytohaemagglutinin from Ricinus communis is subjected to frontal chromatography on Sepharose 4B in the presence of different concentrations of d-galactose, the results illustrating some of the difficulties and limitations that are likely to be encountered in quantitative studies of affinity-chromatographic systems.

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.

HEPARIN INDEPENDENT PURIFICATION OF ANTITHROMBIN III (AT III) BY IMMUNO-AFFINITY CHROMATOGRAPHY RESULTING IN A FUNCTIONALLY INTACT MOLECULE

1987 ◽  
Author(s):  
M Jørgensen
Keyword(s):  
Affinity Chromatography ◽  
Gel Electrophoresis ◽  
Binding Capacity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Single Step ◽  
Heparin Binding ◽  
Purification Method ◽  
Step Procedure ◽  
At Iii ◽  
Sepharose 4B

Previous methods for purification of AT III are based on its heparin-binding capacity. However, in congenital AT III deficiency abnormal inhibitor molecules with impaired binding of heparin and/or thrombin has been reported. The aim of the present study was to develop a purification method based on immuno-affinity chromatography, and thus independent of the heparin binding capacity.Rabbits were immunized with human AT III purified by a three-step procedure involving dextran sulphate precipitation, affinity chromatography on heparin-Sepharose and ion-exchange chromatography on DEAE-Sephadex A-50. Rabbit immunoglobulins against human AT III were isolated by affinity chromatography using purified human AT III coupled to CNBr-activated Sepharose 4B. Trace amounts of immunoglobulin against human albumin, IgG and IgM were removed by solid phase immunoadsorption. The highly purified immunoglobulins against human AT III were coupled to CNBr-activated Sepharose 4B. This anti-AT III-Sepharose was used for single-step purification of AT III from plasma. Elution was performed by Na-citrate buffer at pH 3.0 and the eluted fractions immediately neutralized. The recovery was more than 60%.The purified AT III appeared as a single protein band in SDS-poly-acrylamide gel electrophoresis with or without reduction. Affinity purified AT III and AT III purified by the three-step procedure were indistinguishable when analyzed by crossed immunoelectrophoresis in the absence and the presence of heparin isoelectrical focusing in polyacrylamid gel at a pH 4-6.5 gradient, and SDS-polyacrylamide gel electrophoresis. AT III antigen concentration was determined by electroimmunoassay and the reactive site concentration determined by titration with purified human thrombin using Phe-Pip-Arg-Nan (S-2238) as substrate. The ratio (active site conc.)/(antigen conc.) was identical in the two AT III preparations. It is concluded that this single-step immuno-affinity chromatography gives a high recovery from plasma of a highly purified functionally intact AT III molecule. The purification method is independent of the heparin binding capacity of AT III. This is of particular importance for the purification and characterization of abnormal AT III molecules with impaired heparin-binding site.

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.

Total lactate dehydrogenase and its isoenzymes in serum in the presence of penicillamine and other sulfhydryl compounds.

1977 ◽  
Vol 23 (2) ◽  
pp. 229-233 ◽  
Author(s):  
L L Gershbein ◽  
K G Raikoff
Keyword(s):  
Lactate Dehydrogenase ◽  
Disc Electrophoresis ◽  
The Other ◽  
Rat Serum ◽  
Initial Concentration ◽  
Ldh Isoenzymes ◽  
Allosteric Effectors ◽  
Sulfhydryl Compounds

Abstract Toward delineation of changes in total lactate dehydrogenase (LDH) and in the distribution of LDH isoenzymes as assessed by polyacrylamide disc electrophoresis, we inbucated human and rat sera with various agents, notably sulfhydryl compounds. Although artefacts were apparent when these agents were used without preliminary adjustment of pH, we saw little alteration in total unitage when one or two volumes of serum was mixed with one volume of any of several thiols, especially penicillamine, at an initial concentration of 0.4 mol/liter and pH 7.0-7.5. Under these conditions, penicillamine caused a loss in LDH-5 after incubation for 1 h at 25 degrees C together with small decreases in mobility of the other four isoenzymes toward the anode. A zymosan region appeared below the albumin and tracking dye area. With longer periods of incubation of rat serum with penicillamine or alpha-mercaptosuccinate, a novel band in the zymogram was noted just above the LDH-4 peak. The observations are discussed in terms of allosteric effectors.

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