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 n Revision of enzymology n Evaluation of activity of enzymes in bilogic samples n LDH isoenzymes assay

3. The basic characteristics of the enzymes A E B

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

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

9. Enzymes  proteins n Most biological enzymes are proteins. They speed up chemical reactions in biological systems. (the exception is catalytic RNA).enzymes proteins n 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 substratesactive site

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

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

12. Genetics

13. n Genetic polymorfism multigene diseases Genetic polymorfism multigene diseases n Rare alleles (mutation) hereditary enzymopathies Rare alleles (mutation) hereditary enzymopathies n Consequences: alterations of structure and/or concentration

14. Metabolic consequences A E B C A B E

15. Clinical medicine

17. Enzymes in blood plasma n Functional plasmatic enzymes enzymes of blood clotting, lipoprotein lipaze, ceruloplasmin n Non-functional plasmatic enzymes 1.enzymes from exocrine glands (amylase) 2.intracellular enzymes

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

19. Liver, kidney Inhibitors

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

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

24. LDH - tetramer n H unit and M unit n LDH 1 HHHH heart, brain, kidney n LDH 2 HHHM heart n LDH 3 HHMM smooth muscle n LDH 4 HMMM skeletal muscle n LDH 5 MMMM skeletal muscle, liver

25. LDH - tetramer n H unit aerobic metabolism lactat pyruvate n M unit anaerobic metabolism pyruvate lactat

26. LDH izoenzymes n 1. Heat deactivation - LDH 5 is termo- instable when heated to 57  C n 2. afinity to hydroxybutyrat - myocardial fraction (LDH 1 LDH 2 ) catalyze hydroxybutyrat dehydrogenation LD/HBD low = myocardial affection, LD/HBD high = hepatal affection

27. LDH n Lactat dehydrogenase: hemolysis causes false increase of LDH

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

29. Normal levels of LDH isoenzymes

30. Automatic pipette

31. The gel pouring

32. Agarose gel

33. The sample loading

34. Agarose gel

35. The Sample loading

36. The sample loading

38. Gel with samples in elfo tank

39. Electrophoresis

40. Paper bridges in elfo tank

41. Visualization

42. Line and peak detection

43. Densitometric evaluation

44. Normal levels of LDH isoenzymes

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

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

48. Matrixmetalloproteinasy- zymografie n Elektrophoresis - PolyAacrylamidGelElectrophoresis with gelatine. n Coomasie blue staining

49. Zymogram pro MMP-2 MMP-2

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

51. Multifactorial diseases n Atherosclerosis n Diabetes mellitus n Allergy n Tumors Back

52. Hereditary enzymopathies n Fenylketonuria n Alkaptonuria n Thesaurismosy: glykogenozy, mukopolysacharidozy, glykosfingolipidozy Back