Questions to study.
1. Modern views on intracellular degradation of proteins. Proteolytic enzymes of the cell. Classes of proteolytic enzymes.
2. Intracellular pool of free amino acids and its sources.
3. Common pathways of amino acid catabolism (transamination, deami-nation and decarboxylation).
4. Oxidative deamination. Indirect oxidative deamination.
5. The fate of carbon skeletons of amino acids.
6. Glycogenic and ketogenic amino acids.
Assignment for self-instruction
| | Guidelines for performing the task |
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| Characterize digestion of proteins in gastrointestinal tract | 1. Describe chemical composition of gastric, pancreatic and intestinal juices. 2. Characterize the substrate specificity and optimal pH for gastrointestinal proteases, mechanism of their activation |
| Study digestion of proteins in the small intestine and absorption products of hydrolysis | 1. Name the conditions providing for digestion of proteins in the small intestine. 2. Name proteolytic enzymes of pancreatic and intestinal juice and describe their role in digestion of proteins and peptides |
Ending of the table
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| Study putrefaction of proteins in the large intestine | 1. Define the term putrefaction of proteins in the large intestine. 2. Give examples of conversion of amino acids to toxic products and their detoxification. 4. Write down structural formulas of PAPS and UDP-glucuronic acid and examples of detoxication reactions using these compounds |
| Study the processes of transamination, deamination and decarboxylation of amino acids | 1. Write down the general scheme of transamination of amino acids. 2. Write down the formula of pyridoxal phosphate and molecular mechanism of transamination. 3. Explain the biological significance of transamination and its role in relationship between metabolism of amino acids and metabolism of carbohydrates, lipids, TCA cycle. 4. Name glucogenic and ketogenic amino acids. 5. Describe the types of deamination in living systems. 6. Write down a reaction catalyzed by glutamate dehydrogenase. 7. Write down reactins of indirect deamination of alanine, aspartate, serine. 8. Write down reactions of decarboxylation of histidine, tryptophan, lysine, glutamate. Describe the biochemical and physiological effects of the resulting biogenic amines. 9. Write down a scheme of inactivation of amines in the body |
Library-research paper
1. Cathepsins: their structure, classification, and significance.
2. Role of proteasomes in tissue proteolysis.
LABORATORY WORK
1. Determination of alanine aminotransferase (ALT) in the blood serum
It catalyzes the transfer of an amino group from alanine to α-ketoglutarate to yield pyruvate and glutamate.
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The rate of pyruvate formation is determined by the reaction of pyruvic acid with 2,4-dinitrophenyl hydrazine (DNPH) in an alkaline medium. Addition of 2,4-dinitrophenyl hydrazine results in formation of hydrazone complex with pyruvate. A characteristic color appeares. The intensity of color is related to enzyme activity.
Procedure
► 0.5 ml of substrate solution and 0.1 ml of blood serum are placed into a test tube. It is shaken intensively and placed in thermostat at a temperature of 37 °С for 30 min. After that 0.5 ml of 2,4-dinitro-phenylhydrazine is added. The test tube is shaken intensively and left at room temperature for 20 min. Then 5 ml of 0.4 N NaOH is added, and in 10 min light absorbance is determined using a photocolorimeter at 500-560 nm (green filter) in a cuvette 10 mm wide against the control sample. In preparation of the control sample distilled water is used instead of the blood serum.