1. Participation at practicals 100% and performation of all experiments with presentation of protocols. In case of absence (max. 3 times) at a practical lesson it is necessary to present the topic in the form of seminar work next week or in the credit week.

2. To pass successfully 2 written tests (with minimal evaluation of mark E).

3. Average mark of tests (written in the begining of every lesson) increases the percentage gain of students.

Student is able to explain the basic principles of enzyme catalysed reaction, of bioenergetics, of metabolic regulation at level of cell and animal organism, respectively. He is able te read metabolic map from saccharide, lipid and steroid, protein and amino acid metabolism point of view. He knows metabolism specifics at subcellular, cellular and organ levels in animal organism.

Enzymology.

Enzyme kinetics.

Inhibitors.

Coenzymes.

Bioenergetic metabolism.

Metabolic regulation.

Hormonal regulation.

Tricarboxylic acid cycle.

Carbohydrate metabolism.

Lipid metabolism.

Metabolism of eicosanoids.

Biosynthesis of cholesterol, steroid hormones, bile acids and vitamin D.

Lipoprotein metabolism.

Metabolism of ketone bodies.

Protein degradation and metabolism of amino acids.

Urea cycle.

Biogenic amines.

Biochemistry of digestion.

Biochemistry of liver.

Biochemistry of blood.

Heme biosynthesis and heme degradation.

Biochemistry of kidneys and acid-base balance.

Biochemistry of muscle.

Mechanism of muscle contraction.

Biochemistry of connective tissue.

Calcium metabolism.

Signal transmission in the CNS, synaptic signal transmission.

BIOCHEMISTRY LECTURE SYLLABUS

1. Enzymology.

Enzymes, enzymatic activity, reaction and substrate specificity, enzyme classes.

Catalytic function of enzymes and active sites; activation energy.

Enzyme catalysis, uncatalyzed reaction, enzyme-catalyzed reaction, principles of enzyme catalysis (serine proteinases as example).

Localization of enzymes in cell (cytoplazma, membrane, mitochondrion).

Isoenzymes. Lactate dehydrogenase as an example.

2. Enzyme kinetics.

Michaelis-Menten kinetics, isosteric and allosteric enzymes.

Kinetic constants Vmax, Km, kcat and C.

Inhibitors.

Types of inhibitions (reversible and irreversible; competitive, uncompetitive and non-competitive, allosteric).

3. Coenzymes.

Redox coenzymes: NAD+, NADP+, FMN, FAD, ubiquinone, ascorbic acid, lipoamid, iron-sulfur cluster, heme.

Group-transferring coenzymes: nucleoside phosphates, coenzyme A, TPP, PLP, biotin, THF, cobalamin.

Activated metabolites (UDPglucose, CDPcholine, PAPS, SAM).

4. Bioenergetic metabolism.

Endergonic and exergonic reactions. Energetic coupling.

ATP as a general energy transporter (structure, types of ATP formation).

Respiratory chain, its localization and enzyme systems.

Electrochemical gradient and protonmotive power.

ATP synthesis (oxidative phosphorylation).

5. Metabolic regulation.

Regulatory mechanisms.

Allosteric regulation. Transcription control. Hormonal control.

Endocrine, paracrine and autocrine hormone effects.

Lipophilic hormones, steroid hormones and iodothyronines.

Mechanism of action of lipophilic hormones, steroid receptors.

Hydrophilic hormones, hormones derived from amino acids, peptide hormones, proteohormones.

Mechanism of action of hydrophilic hormones, second messengers.

6. Carbohydrate metabolism.

Glycolysis (reactions, energy profile, aerobic and anaerobic oxidation of glucose).

Oxoacid dehydrogenases – PDH reaction.

Tricarboxylic acid cycle (reactions, functions, regulation).

Pentose phosphate pathway of glucose degradation.

Gluconeogenesis.

Biochemistry of liver.

Functions of the liver.

Hepatic metabolism: carbohydrate, lipid, amino acid and protein metabolism.

Biotransformations, ethanol metabolism.

7. Carbohydrate metabolism.

Glycogen metabolism (glycogenesis, glycogenolysis).

Regulation of carbohydrate metabolism.

Lipid metabolism.

Fatty acid degradation (fatty acid transport, β-oxidation).

Degradation of unsaturated fatty acids and oddnumbered fatty acids.

α-oxidation, ω-oxidation, degradation of unusually long fatty acids.

8. Lipid metabolism.

Fatty acid synthesis.

Biosynthesis of triacylgylcerols and phospholipids.

Metabolism of eicosanoids.

Biosynthesis of cholesterol and its interconversions.

Lipoprotein metabolism.

Metabolism of ketone bodies.

9. Protein degradation and metabolism of amino acids.

Proteolysis, proteolytic enzymes, proteasome.

Amino acid degradation (transamination, oxidative deamination).

Glucogenic and ketogenic amino acids.

Urea cycle.

Amino acid decarboxylation, synthesis of biogenic amines.

Biosynthesis of non-essential amino acids.

Essential, non-essential amino acids, limiting amino acid.

Biosynthesis of glutamate, serine, aspartate and cysteine families.

Glutathione – γ-glutamyl cycle.

10. Biochemistry of digestion.

Nutrients, organic substances, vitamins, minerals and trace elements.

Hydrolysis and resorption of food components.

Digestive juices.

Digestion and resorption.

11. Biochemistry of blood.

Blood: composition and functions.

Plasma proteins, lipoproteins.

Heme biosynthesis and heme degradation.

Hemoglobin, iron metabolism, gas transport, erythrocyte metabolism.

Acid-base balance.

12. Biochemistry of kidneys and acid-base balance.

Functions of the kidneys.

Urine formation: ultrafiltration, resorption, secretion, clearance.

Organic and anorganic components of urine.

Functions in the acid-base balance: proton and ammonia excretion.

Electrolyte and water recycling: calcium and phosphate ions, sodium ions, water.

Renal hormones: calcitriol, erythropoietin, renin-angiotensin system.

Biochemistry of muscle.

Mechanism of muscle contraction.

Control of muscle contraction.

Energy metabolism in the white and red muscle fibers, creatine metabolism.

Cori and alanine cycle.

13. Biochemistry of connective tissue.

Bone and teeth.

Calcium metabolism.

Collagens. Extracellular matrix.

Biochemistry of nerve system.

Signal transmission in the CNS, synaptic signal transmission.

Resting and action potential.

Neurotransmitters: acetylcholine, biogenic amines, peptides, purine derivatives, catecholamines.

Receptors for neurotransmitters (ionotropic, metabotropic), acetylcholine receptors (nicotinic, muscarinic).

PRACTICAL LESSON PLAN

1.Safety rules for working in biochemical laboratory on practical lessons.

Organization of the course in Biochemistry.

Kinetics of enzyme-catalyzed reactions. Temperature dependence of enzyme reaction

Temperature dependence of the trypsin-catalyzed (EC 3.4.21.4) hydrolysis of N-α-benzoyl-DL-arginyl-p-nitroanilide (BAPA).

2. Kinetics of enzyme-catalyzed reactions. pH dependence of enzyme reactions.

Determination of pH optimum for the trypsin-catalyzed hydrolysis of BAPA.

3. Kinetics of enzyme-catalyzed reactions. Substrate concentration dependence of reaction rate.

a) The influence of BAPA concentration on the rate of trypsin-catalyzed hydrolysis.

b) Determination of kinetic constants: Michaelis constant Km (Lineweaver-Burk), catalytic constatnt kcat and overall constant C.

4. Inhibition of enzyme activity.

a) The influence of blood serum inhibitor on the rate of trypsin-catalyzed hydrolysis of BAPA.

b) The influence of α1-antitrypsin on the trypsin-catalyzed hydrolysis of α-tosyl-L-arginyl-p-nitroanilide (tutorial).

5. I. Written credit test: Enzymology and bioenergetics.

Activation of enzyme activity.

The influence of calcium cations on the rate of trypsin-catalyzed hydrolysis of BAPA.

6. Carbohydrate metabolism.

Determination of glucose concentration in blood of animals.

Determination of total lactate dehydrogenase (LDH, EC 1.1.1.27) activity in blood serum of domestic animals.

7. Polysaccharide metabolism.

Determination of α-amylase (AMS, EC 3.2.1.1) activity in blood serum and urine of domestic animals.

Lipid metabolism.

Determination of triacylglycerol concentration in blood serum of animals.

Determination of cholesterol concentration in blood serum of domestic animals.

8. Determination of proteins in sera of domestic animals.

a) Quantitative determination of total proteins in blood serum of animals by biuret method.

b) Determination of albumin concentration in blood serum of animals.

c) Salting out of serum proteins using ammonium sulfate.

d) Electrophoresis of blood plasma proteins.

9. Amino acid metabolism – transamination reactions.

Determination of aspartate aminotransferase (AST, EC 2.6.1.1) activity in blood serum of domestic animals.

10. II. Written credit test: Carbohydrate and lipid metabolism.

Biochemistry of liver.

Determination of γ-glutamyl transferase (GGT, EC 2.3.2.2) activity in blood serum of domestic animals.

11. Biochemistry of blood.

a) Determination of hemoglobin concentration in blood of animals.

b) Determination of iron concentration in blood serum of domestic animals.

12. Biochemistry of kidney.

Determination of urea concentration in blood serum of domestic animals by Berthelot method.

13. Biochemistry of muscle contraction.

Determination of creatine kinase (CK, EC 2.7.3.2) activity in blood serum of animals.

Biochemistry of bone.

Determination of alkaline phosphatase (ALP, EC 3.1.3.1) activity in blood serum of domestic animals.

Student presentations of their seminary works.

Awarding credit points to students.

Harvey, R.A., Ferrier, D.R.: Lippincott´s Illustrated Reviews: Biochemistry. 5th Edition. Baltimore, Lippincott Wiliams and Wilkins, 2011, 521pp.

Koolman, J., Roehm, K. H.: Color Atlas of Biochemistry. 2nd Edition. Stuttgart; New York : Georg Thieme Verlag, 2005, 476 pp.

Nelson, D. L., Cox, M. M.: Lehninger Principles of Biochemistry. 4th Edition. New York : W. H. Freeman and Company, 2005, 1119 pp.

Stryer, L.: Biochemistry. 3rd Edition. New York : W. H. Freeman and Company, 1988, 1089 pp.

Kostecká, Z., Heinová, D., Sobeková, A., Milkovičová, M., Šimková, J.: Practical Course in Biochemistry for Joint Study Programme Animal Science, Košice: UVMP, 2017

Continuous assessment:

continuous tests (on practical lessons from actually tought topics), solution of tasks (in protocol form of performed practical work on laboratory lessons), two credit written tests from explained chapters

Conditions for completion of course:

1. Participation at practicals 100% and performation of all experiments with presentation of protocols. In case of absence (max. 3 times) at a practical lesson it is necessary to present the topic in the form of seminar work next week or in the credit week.

2. To pass successfully 2 written tests (with minimal evaluation of mark E).

3. Average mark of tests (written in the begining of every lesson) increases the percentage gain of students.

Final assessment:

final exam (written and oral)