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The separation of lactatdehydrogense (LDH) isoenzymes by agarose electrophoresis

The separation of lactatdehydrogense (LDH) isoenzymes by agarose electrophoresis. MUDr. Michal Jurajda Svatava Tschöplová Šárka Kuchtíčková Pavla Součková. The objective s of the practical training. Revision of enzymology Evaluation of activity of enzymes in bilogic samples

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The separation of lactatdehydrogense (LDH) isoenzymes by agarose electrophoresis

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  1. The separation of lactatdehydrogense (LDH) isoenzymes by agarose electrophoresis MUDr. Michal Jurajda Svatava Tschöplová Šárka Kuchtíčková Pavla Součková

  2. The objectives of the practical training • Revision of enzymology • Evaluation of activity of enzymes in bilogic samples • LDH isoenzymes assay

  3. E A B The basic characteristics of the enzymes

  4. Enzymes = biocatalyzers • Enzymes Lower the Activation Energy of Reactions • Speed up chemical reactions • Equilibrium is not influenced • Michaelis-Menten Equation • Km is defined as the [S] that results in half-maximal rate.

  5. Units • Catal is the unit of enzymatic activity 1cat =activity which converts 1mol of substrate to product during 1second • Katal characterizes amount of enzyme not properties of that (Km)

  6. Enzymes  proteins • Most biological enzymes are proteins . They speed up chemical reactions in biological systems. (the exception is catalytic RNA). • The segment of the enzyme molecule that does the work is called the active site . The amino-acid residues in this site are arranged in specific 3D conformation enabling interaction with substrates

  7. Izoenzymes • They catalyze the same reaction but they are different in the structure  physical-chemical characteristics • Primarny - different genes • Secondary - one gene produce different enzymes by different posttranslation alterations (acetylation, cleavage)

  8. Regulation of enzymatic activity in the biological systems • Regulation of transcription • Activation of proenzymes • Inhibition by specific inhibitors (competitive, non-competitive) • Metabolic pathways

  9. Genetics

  10. Genetic polymorfismmultigene diseases • Rare alleles (mutation)hereditary enzymopathies • Consequences:alterations of structure and/or concentration

  11. Metabolic consequences E E A B A B C

  12. Clinical medicine

  13. Enzymes in blood plasma • Functional plasmatic enzymesenzymes of blood clotting, lipoprotein lipaze, ceruloplasmin • Non-functional plasmatic enzymes1.enzymes from exocrine glands (amylase)2.intracellular enzymes

  14. The concentration of enzymes in the blood plasma • The level of enzymatic activity of individual enzymes in the cell • The localization of the enzyme in the cell • The extent of cellular damage • The number of damaged cells • The elimination rate of the enzyme

  15. Inhibitors Inhibitors Liver, kidney

  16. Diagnostics • CK creatinkinase, CK-MB myocardial band • AST aspartate aminotransferase (mit.) • ALT alaninaminotransferase • LDH laktatedehydrogenase

  17. The separation of isoenzymes • Electrophoresis or chromatography • Activity assay under different conditions pH, temperature, different substrates

  18. LDH - tetramer • H unit and M unit • LDH1 HHHH heart, brain, kidney • LDH2 HHHM heart • LDH3 HHMM smooth muscle • LDH4 HMMM skeletal muscle • LDH5 MMMM skeletal muscle, liver

  19. LDH - tetramer • H unit aerobic metabolismlactat pyruvate • M unit anaerobic metabolismpyruvate lactat

  20. LDH izoenzymes • 1. Heat deactivation - LDH5 is termo-instable when heated to 57C • 2. afinity to hydroxybutyrat - myocardial fraction (LDH1 LDH2 ) catalyze hydroxybutyrat dehydrogenation LD/HBD low = myocardial affection, LD/HBD high = hepatal affection

  21. LDH • Lactat dehydrogenase: hemolysis causes false increase of LDH

  22. Electrophoretic separation of LDH in agarose gel • Agarose in barbital buffer • Visualization: • 1. lithium lactat • 2. p-iodonitroterazoluim violet - colour substantion, blue when reduced • 3. NAD+ • 4. KCN • 5. Fanezinmethosulfat electron transducer from NADH • 5% acetic acid

  23. Normal levels of LDH isoenzymes

  24. Automatic pipette

  25. The gel pouring

  26. Agarose gel

  27. The sample loading

  28. Agarose gel

  29. The Sample loading

  30. The sample loading

  31. The sample loading

  32. Gel with samples in elfo tank

  33. Electrophoresis

  34. Paper bridges in elfo tank

  35. Visualization

  36. Line and peak detection

  37. Densitometric evaluation

  38. Normal levels of LDH isoenzymes

  39. Matrixmetalloproteinases • enzymes capable to cleave ECM • release of growth and motility factors from ECM • activity regulation (transkription, plasmin) • tissue inhibitors of MMPs (TIMPs)

  40. Matrixmetalloproteinases-zymography • SDS elektrophoresis - SDS coats proteins and form polyanionts, elektrophoresis runs toward anode. • SDS is removed with Triton and proteins renaturate their enzymatic activity is restored.

  41. Matrixmetalloproteinasy-zymografie • Elektrophoresis - PolyAacrylamidGelElectrophoresis with gelatine. • Coomasie blue staining

  42. Zymogram pro MMP-2 MMP-2

  43. Sources • http://esg-www.mit.edu:8001/esgbio/7001main.html • Biochemie v obrazech, J. Musil, O.Nováková, Avicenum 1990 • Enzymologie jaterních nemocí, J. Pojer, SZN 1968 • Enzymologie srdečního infarktu, J. Pojer, SZN 1963

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