Lactate dehydrogenase characteristics

Suaud-Chagny and Gonon [3] presented a new procedure for protein immobilization adapted to carbon microelectrode characteristics. The principle of this method of immobilization is based on the association of the protein with an inert porous film immobilized around the active tip of the electrode. For this purpose the carbon was coated with an inert, electrochemically obtained protein sheath (bovine serum albumin, BSA) a few micrometers thick. Then the sheath around the fiber was impregnated with lactate dehydrogenase (LDH), which could be immobilized onto the electrode and resulted in an electrode sensitive to pyruvate. 

Part—I has three chapters that exclusively deal with General Aspects of pharmaceutical analysis. Chapter 1 focuses on the pharmaceutical chemicals and their respective purity and management. Critical information with regard to description of the finished product, sampling procedures, bioavailability, identification tests, physical constants and miscellaneous characteristics, such as ash values, loss on drying, clarity and color of solution, specific tests, limit tests of metallic and non-metallic impurities, limits of moisture content, volatile and non-volatile matter and lastly residue on ignition have also been dealt with. Each section provides adequate procedural details supported by ample typical examples from the Official Compendia. Chapter 2 embraces the theory and technique of quantitative analysis with specific emphasis on volumetric analysis, volumetric apparatus, their specifications, standardization and utility. It also includes biomedical analytical chemistry, colorimetric assays, theory and assay of biochemicals, such as urea, bilirubin, cholesterol and enzymatic assays, such as alkaline phosphatase, lactate dehydrogenase, salient features of radioimmunoassay and automated methods of chemical analysis. Chapter 3 provides special emphasis on errors in pharmaceutical analysis and their statistical validation. The first aspect is related to errors in pharmaceutical analysis and embodies classification of errors, accuracy, precision and makes... 

Figure 8.10 The quaternary structure of proteins. The enzyme lactate dehydrogenase (EC 1.1.1.27) has a relative molecular mass of approximately 140 000 and occurs as a tetramer produced by the association of two different globular proteins (A and B), a characteristic that results in five different hybrid forms of the active enzyme. The A and B peptides are enzymically inactive and are often indicated by M (muscle) and H (heart). The A4 tetramer predominates in skeletal muscle while the B4 form predominates in heart muscle but all tissues show most types in varying amounts.

The characteristic stereospecificity of enzymes has been exploited in the design of an electrochemical cell for the conversion of L-lactate to D-lactate (Scheme 23)117. Enzymatic oxidation of L-lactate by L-lactate dehydrogenase affords pyruvate. Pyruvate is then reduced electrochemically to racemic lactate. A second enzymatic oxidation of the latter by L-lactate dehydrogenase selectively converts L-lactate to pyruvate, leaving D-lactate behind. The ingenious feature of this system is the fact that pyruvate can be re-reduced... 

An example of an enzyme which has different isoenzyme forms is lactate dehydrogenase (LDH) which catalyzes the reversible conversion of pyruvate into lactate in the presence of the coenzyme NADH (see above). LDH is a tetramer of two different types of subunits, called H and M, which have small differences in amino acid sequence. The two subunits can combine randomly with each other, forming five isoenzymes that have the compositions H4, H3M, H2M2, HM3 and M4. The five isoenzymes can be resolved electrophoretically (see Topic B8). M subunits predominate in skeletal muscle and liver, whereas H subunits predominate in the heart. H4 and H3M isoenzymes are found predominantly in the heart and red blood cells H2M2 is found predominantly in the brain and kidney while HM3 and M4 are found predominantly in the liver and skeletal muscle. Thus, the isoenzyme pattern is characteristic of a particular tissue, a factor which is of immense diagnostic importance in medicine. Myocardial infarction, infectious hepatitis and muscle diseases involve cell death of the affected tissue, with release of the cell contents into the blood. As LDH is a soluble, cytosolic protein it is readily released in these conditions. Under normal circumstances there is little LDH in the blood. Therefore the pattern of LDH isoenzymes in the blood is indicative of the tissue that released the isoenzymes and so can be used to diagnose a condition, such as a myocardial infarction, and to monitor the progress of treatment. 

Cytochrome 62 is stereospecific for l(- -)-lactate. It also oxidizes other a-hydroxymonocarboxylic acids at slow rates 80, 96). As electron acceptors ferricyanide, methylene blue, 2,6-dichloroindophenol, 1,2-naphthoquinone 4-sulfonate, and cytochrome c have been used. This wide acceptor specificity is characteristic of a number of flavoproteins, which are generally capable of reducing quinoid structures and ferric compounds 97). However, as will be seen below, cytochrome c is considered to be the physiological electron acceptor for the yeast L-lactate dehydrogenase. 

Tissues contain characteristic isozymes or mixtures of isozymes. Enzymes such as lactate dehydrogenase and creatine kinase differ from one tissue to another. 

The diagnosis of organ disease is aided by measurement of a number of enzymes characteristic of that tissue or organ. Most tissues have characteristic enzyme patterns (Table 8-2) that may be reflected in the relative serum concentrations of the respective enzymes in disease. The diseased tissue can be further identified by determination of the isoenzyme pattern of one of these enzymes (e.g., lactate dehydrogenase, creatine kinase) in the serum, since many tissues have characteristic isoenzyme distribution patterns for a given enzyme. For example, creatine kinase (CK) is a dimer composed of two subunits, M (for muscle) and B (for brain), that occur in three isoenzyme forms, BB(CKi), MB(CK2) and MM(CK3), which catalyze the reversible phosphorylation of creatine with adenosine triphosphate (ATP) as the phosphate donor ... 

Interruption of the heart s blood supply leads to the death of cardiac muscle cells. The symptoms of myocardial infarction include pain in the left side of the chest that may radiate to the neck, left shoulder, and arm, and irregular breathing. The initial diagnosis is based on these and other symptoms. Therapy is instituted immediately. Physicians then use several enzyme assays to confirm the diagnosis and to monitor the course of treatment. The enzymes most commonly assayed are creatine kinase (CK) and lactate dehydrogenase (LDH). Each enzyme s activity shows a characteristic time profile in terms of its release from damaged cardiac muscle cells and rate of clearance from blood (Figure 6A). 

Fig. 1. Time course of cell death parameters in tumor necrosis factor (TNF)-induced apoptosis. Human hepatoma cells (HepG2) were incubated in the presence of 400 nM actinomycin D plus 1.6 ng/ml recombinant human TNF (rhTNF)-a. MTT-dye reduction (parameter of mitochondrial function), lactate dehydrogenase (LDH) release (parameter for cell membrane rupture), and DNA fragmentation (parameter associated with typically apoptotic nuclear changes) were determined over a time period of 24 h. As is characteristic of apoptotic cell death, DNA fragmentation always preceded the other death parameters. Similar findings have been obtained in primary hepatocyte cultures and murine livers...

The growth of microorganisms may have an important effect on the flavour of food. An example is the lactic acid bacteria Lactococcus lactis, which produce mainly lactate providing a characteristic acidic flavour and contributing to the preservation of fermented food. In an innovative approach, in situ 13C NMR was used to investigate glycolysis by an L. lactis strain deficient in lactate dehydrogenase.272... 

Myocardial infarction occurs when the blood supply to the heart muscle is blocked for an extended time. If this lack of blood supply, called ischemia, is prolonged, the myocardium suffers irreversible cell damage and muscle death, or infarction. When this happens, the concentration of cardiac enzymes in the blood rises dramatically as the dead cells release their contents into the bloodstream. Although many enzymes are liberated, three are of prime importance. These three enzymes, creatine phosphokinase (CPK), lactate dehydrogenase (LDH), and aspartate aminotransferase/serum glutamate-oxaloacetate transaminase (AST/SGOT), show a characteristic sequential rise in blood serum level following myocardial infarction and then return to normal. This enzyme profile, shown in the ac-... 

Despite the fact that they can be much more reactive than nitrenes, carbene derivatives are less frequently cited in reports on photoaffinity labels. Two such derivatives of thyroid hormone have been described in which the amino group of the alanine side chain of 3,5,3 -triiodo-L-thyronine or 3,5,3, 5 -tetraiodo-L-thyronine was derivatized with 2-diazo-3,3,3-trifluoropropionate (246). Irradiation at 254 nm of these compounds with several cell lines yielded low incorporation of reagent (0.5-13.5%) into two forms of thyroid hormone receptor proteins. Low incorporation was also characteristic of early reports of photoaffinity labeling (e.g., 247) in which 0 -ethyl-2-diazomalonyl-cAMP and A, C)2. (ji(ethyl-2-diazomalonyl)-cAMP were used to generate carbenes which modify rabbit muscle phosphofructokinase. Similarly, only 3-14% of the potential sites of lactate dehydrogenase were labeled by the carbene derived from 3-(3//-diazirino)pyridine adenine dinucleotide (248). 

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