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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 MUDr. Michal Jurajda Svatava Tschöplová Šárka Kuchtíčková Pavla Součková
The objectives of the practical training • Revision of enzymology • Evaluation of activity of enzymes in bilogic samples • LDH isoenzymes assay
E A B The basic characteristics of the enzymes
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.
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)
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
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)
Regulation of enzymatic activity in the biological systems • Regulation of transcription • Activation of proenzymes • Inhibition by specific inhibitors (competitive, non-competitive) • Metabolic pathways
Genetic polymorfismmultigene diseases • Rare alleles (mutation)hereditary enzymopathies • Consequences:alterations of structure and/or concentration
Metabolic consequences E E A B A B C
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
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
Inhibitors Inhibitors Liver, kidney
Diagnostics • CK creatinkinase, CK-MB myocardial band • AST aspartate aminotransferase (mit.) • ALT alaninaminotransferase • LDH laktatedehydrogenase
The separation of isoenzymes • Electrophoresis or chromatography • Activity assay under different conditions pH, temperature, different substrates
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
LDH - tetramer • H unit aerobic metabolismlactat pyruvate • M unit anaerobic metabolismpyruvate lactat
LDH izoenzymes • 1. Heat deactivation - LDH5 is termo-instable when heated to 57C • 2. afinity to hydroxybutyrat - myocardial fraction (LDH1 LDH2 ) catalyze hydroxybutyrat dehydrogenation LD/HBD low = myocardial affection, LD/HBD high = hepatal affection
LDH • Lactat dehydrogenase: hemolysis causes false increase of LDH
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
Matrixmetalloproteinases • enzymes capable to cleave ECM • release of growth and motility factors from ECM • activity regulation (transkription, plasmin) • tissue inhibitors of MMPs (TIMPs)
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.
Matrixmetalloproteinasy-zymografie • Elektrophoresis - PolyAacrylamidGelElectrophoresis with gelatine. • Coomasie blue staining
Zymogram pro MMP-2 MMP-2
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