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Lesson 9. BIOLOGICAL OXIDATION. OXIDATIVE DECARBOXYLATION OF PYRUVATE. CITRIC ACID CYCLE. TISSUE RESPIRATION AND OXIDATIVE PHOSPHORYLATION

Questions to study.

1. Metabolism and energy as the most important features of living creatures. Anabolism and catabolism, their general characteristics and relationship. Central metabolic pathways. Stages of catabolism.

2. Acetyl CoA: its structure, significance and pathway of its synthesis.

3. Citric acid cycle: partial reactions, regulation and biological significance.

4. Tissue respiration. Structure of respiratory chain of mitochondria.

Assignment for self-instruction

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Task

Guidelines for performing the task

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Recall the main points of bioenergetics

1. Write down classification of organisms according to energy sources, type of nutrition and oxygen requirement.

2. Define the term high-energy compounds

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Study the structure and functions of the high-energy compounds

1. Write down formulas of the following high-energy compounds:

а) nucleoside triphosphates;

б) acyl phosphates anhydrides of carboxylic and phosphoric acids;

в) enol phosphate - phosphoenolpyruvate;

г) guanidine phosphates - creatine phosphate;

д) thiol esters - acetyl CoA and succinyl CoA;

е) sulfonium compounds - S-adenosyl methionine.

2. Explain the biological significance of high-energy compounds.

3. Describe the key role of ATP in energy metabolism as a universal source of an energy (universal energy currency)

Continued of the table

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Study the structure and function of enzymes taking part in the processes of biological oxidation

1. Recall the structure and function of pyridinic and flavine-linked dehydrogenases.

2. Write down formulas of NAD+, NADP+, FMN, FAD

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Study the common ways of nutrient catab-olism and unification (simplifying) of energy of substrates

1. Write down the energy value (in calories) of oxidation for 1 g of proteins, 1 g of lipids and 1 g of carbohydrates.

2. Make a schematic representation of the stages of unification of dietary compounds in the human body

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Study the tricarboxylic acid cycle (TCA cycle)

1. Write down the reactions of TCA cycle.

2. Study the mechanisms regulating TCA cycle. Write the regulatory enzymes and list the factors that influence the TCA cycle rate.

3. Define the term substrate-level phosphorylation. Write the reaction of substrate-level phosphorylation that takes place in TCA cycle

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Study the history of development of the concept of respiration and biological oxidation

1. Describe the hypotheses and theories by Lavoisier, Sheynbayn, Bach, Palladin, Wieland.

2. Describe the modern consept of tissue oxidation and oxidative phosphorylation

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Study the mechanism of mitochondrial electron transport chain (ETC)

function and its connection with ATP synthesis

1. Describe the main groups of electron carriers of ETC.

2. Make a schematic representation of the sequence of electron carriers in the respiratory chain.

3. Designate the respiratory complexes, reactions where hydrogen atoms are transported and reactions of transporting only electrons. Formation of endogenous water. Products of incomplete reduction of oxygen in the respiratory chain and protection system.

4. List the inhibitors of tissue respiration. What inhibitors of tissue respiration do you know?

5. Designate the reactions with maximal differences of redox potentials between electron carriers, and write down the amount of released energy (kJ/mol).

6. Make a schematic drawing of mitochondrial structure and ATP-synthase of inner mitochondial membrane.

7. Explain the concept of coupling of tissue respiration and oxidative phosphorylation by P. Mitchell.

8. What is the stoichiometric ratio P/O? Calculate the value of P/O ratio for oxidation of malic and succinic acids.

9. Define the term uncoupling of oxidative phosphorylation, write down examples of uncouplers

Ending of the table

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10. Explain why the TCA cycle is called the main power boiler of the cell? Calculate the amount of reduced coenzymes formed upon oxidation of acetyl CoA and the quantity of ATP that are synthesized in oxidative phosphorylation.

11. Explain biological significance of close connection between TCA cycle and oxidative phosphorylation

Library-research paper

1. Structural organization of mitochondria.

LABORATORY WORK 1. Revealing succinate dehydrogenase in muscle tissue

Succinate dehydrogenase (SDH) catalyzes the oxidation reaction of suc-cinic acid to yield fumaric acids. Flavine adenine dinucleotide (FAD) is co-enzyme of SDH.

The reaction catalyzed by this enzyme may be observed under anaerobic conditions in the presence of methylene blue or sodium 2,6-dichlorophe-nolindophenol (DCPIPNa) as end electron acceptor. Methylene blue and DCPIP Na are chemical compounds used as redox dye. They are acceptors of hydrogen from reduced FAD. Oxidized methylene blue and DCPIP have a blue color, their reduced forms are colorless.

Oxidation of succinate in the reaction is inhibited by malonic acid, which acts as a competitive inhibitor of succinate dehydrogenase. Procedure

► Fresh muscle tissue weighing about 1 gm is minced with scissors and ground in a mortar with a small amount of water (2-3 ml) for 1 min.

Prepared in this way muscle slurry is carried to a double layer of gauze, placed in a funnel, washed with water, transferred to filter paper and dried. Then 3 ml of phosphate buffer (pH 7.4) is poured into 3 test tubes and 0.1 gm muscle slurry is added to each test tube with buffer.

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