Lactate dehydrogenase labelling

Label-free optical detection 199 Lactate dehydrogenase 465 -469, 472 A-repressor folding kinetics 551 Lattice simulations 597 Leaving group 81, 86, 88-93 Levinthal s paradox 575,576, 598, 599,600... 

Raman spectroscopy can offer a number of advantages over traditional cell or tissue analysis techniques used in the field of TE (Table 18.1). Commonly used analytical techniques in TE include the determination of a specific enzyme activity (e.g. lactate dehydrogenase, alkaline phosphatase), the expression of genes (e.g. real-time reverse transcriptase polymerase chain reaction) or proteins (e.g. immunohistochemistry, immunocytochemistry, flow cytometry) relevant to cell behaviour and tissue formation. These techniques require invasive processing steps (enzyme treatment, chemical fixation and/or the use of colorimetric or fluorescent labels) which consequently render these techniques unsuitable for studying live cell culture systems in vitro. Raman spectroscopy can, however, be performed directly on cells/tissue constructs without labels, contrast agents or other sample preparation techniques. 

Figure 8-38. Inhibition of immune precipitation of I-labeled t>-lactate dehydrogenase by increasing amounts of unlabeled purified D-lactate dehydrogenase (O), a 0,1% Triton X-100 extract of wild type membrane vesicles ( ), a 0.1% Triton X-100 extract of mutant membrane vesicles ( ), and 0.1% Triton X-100 in 0.1 Af potassium phosphate buffer, pH 7.1 (A). The 0.1% Triton X-100 extracts of wild type and mutant vesicles contained the same amounts of protein. The inhibitory effect of the wild type extract was compared with that of a purified i>lactate dehydrogenase preparation containing the same number of units of enzyme activity. [From S. Short and H. R. Kaback, J. Biol. Chent. 250 4285 (1975).]...

Other isolated enzymes which it has already proved possible to successfully spin label include carbonic anhydrase, creatine kinase, phosphofructokinase, lactate dehydrogenase, liver alcohol dehydrogenase, lysozyme and ribonuclease, as well as the coenzymes NAD and vitamin B12. 

The most commonly used approaches are the neutral red assay (cell viability and membrane damage), the Lowry (labeled proline), Coomassie blue, and Kenacid blue assays (cell proliferation and total cell protein), the MTT or tetrazolium assay (mitochondrial function), and the intracellular lactate dehydrogenase activity test (cell lysis). 

Label-free hybridization 87 Label-free pyrosequencing 87 LACK antigen 201 Lactate dehydrogenase 1326... 

The use of alkaline phosphatase as an enzyme label allows enhancement of the sensitivity by using phosphoenolpyruvate as substrate and the utilization of a separate detection column in the ET unit for the determination of the product (pyruvate) by substrate recycling. This is accomplished by using the substrate recycling system described above [18] comprising the coimmobilized enzymes lactate dehydrogenase (reduces pyruvate to lactate under the consumption of... 

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). 

When H, = H, H, = H and H = H when H, = H, H, = H and Hi, = H. The (35)-[3- H,2H]- and (3S)-[3-3H, H]oxaloacetates were enzymically synthesized from stereospecifically labeled samples of aspartate using glutamate-oxaloacetaie transaminase. Configurational analysis of the samples of [ H, H H] pyruvate involved enzymic conversion to chiral labeled acetate (lactate dehydrogenase/lactate oxidase), and the sequential use of malate synthase and fiimarase (see text) (223). 

Fig. 2 Lactate dehydrogenase a) a ribbon representation of the tetramer of the B. stearothermophilus enzyme with each peptide chain depicted in a different color. The cofactor and oxamate inhibitor are colored according to atom type, as is fructose bisphosphate. which is an allosteric regulator of the enzyme, b) On the left is a detailed view of the enzyme active site as seen in the crystal structure. The ligand is highlighted in green and key amino acid residues are labeled. This is compared with the traditional two-dimensional representation of the enzyme mechanism on the right. Note that the residue numbers differ slightly from those of the muscle enzyme discussed in the test. (View this an i i color at www.dekker.com.)...

Probably the most fully studied group of isoenzymes is that of lactate dehydrogenase (LDH). The quaternary structure of the enzyme consists of four subunits that are of two different types. One is labeled H because it is the predominant subunit present in the LDH enzyme found in heart muscle cells. The other, labeled M, predominates in other muscle cells. There are five possible ways to combine these four subunits to form the enzyme (see I Figure 10.14). Each combination has slightly different properties, which allows them to be separated and identified by elecfrophoresis. 

Spector and Miller (13) have used the pyruvate kinase/lactate dehydrogenase coupled assay to observe activity at 340 nm, where none of the nucleotides absorbs appreciably. The reaction can also be followed by use of radioactive substrates and separation of the labeled products (4, 11, 16). The choice of buffer can be important, as Tris, glycylglycine, and phosphate (4, 9) inhibit the enzyme. HEPES appears to be the buffer of choice at pH values above 7 (9). Acetate should be used as a counterion when required as chloride, sulfate, and other anions interact with the enzyme (14, 17). 

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