Textbook - BIOLOGICAL CHEMISTRY - Gubsky Yu.I. - 2000

Chapter II. GENERAL LAWS OF METABOLISM

CHAPTER 8. METABOLISM: CATABOLISM, ANABOLISM

8.2. METHODS FOR STUDYING METABOLISM

Various chemical, physicochemical, and physical Methods are used to study METABOLISM, allowing the identification of specific metabolites, the evaluation of their transformations, and their biological significance in carrying out individual physiological Functions of the entire Organism, specialized Tissues (Muscle, nerve, connective, etc.), individual Cells, and subcellular structures.

Modern methods for separating and purifying complex biological mixtures and isolating individual compounds from them include Chromatography—ion-exchange, adsorption, partition, Gel filtration, affinity (biospecific)—and Electrophoresis. To study The Structure of Biomolecules, infrared and ultraviolet spectroscopy, electron paramagnetic Resonance and nuclear magnetic resonance spectroscopy, fluorescence and X-Ray Structural Analysis are employed.

Specific Metabolic pathways are localized within defined intracellular regions—compartments—separated by Introduction/36.html">Biological Membranes, in particular:

- in The Nucleus — reactions of DNA Biosynthesis (Replication), synthesis of messenger and other classes of RNA (Transcription);

- in the Cell/35.html">Mitochondria — reactions of The Tricarboxylic Acid Cycle, fatty acid β-oxidation, electron transport, and Oxidative Phosphorylation;

- in the Cytoplasm (Cytosol) — reactions of Glycolysis, Gluconeogenesis, fatty acid synthesis, Amino acid metabolism, etc.;

- in the Ribosomes — reactions of Protein Synthesis (formation of polypeptide chains);

- in the membranes of The Endoplasmic reticulum — cytochrome P-450-dependent processes of oxidative hydroxylation of hydrophobic substrates of endogenous and exogenous origin ("microsomal oxidation" reactions);

- in the Lysosomes — reactions of hydrolytic Cleavage of Biopolymers—Proteins and Nucleic Acids (both endogenous biomolecules and foreign compounds taken up by certain cells via endocytosis).

To study biochemical reactions and individual metabolites localized in specific cellular compartments, the method of differential centrifugation is used. The Essence of the method lies in sequential centrifugation at various speeds of biological tissue homogenates (Liver, muscle, Brain, etc.) containing suspended subcellular structures (nuclei, mitochondria, lysosomes, various membrane vesicles, ribosomes). To obtain a homogenate, the tissue is disintegrated in a special homogenizer by suspending it in a biologically inert solution (0.25 M sucrose solution, salt solutions). Depending on size and mass, these Organelles sediment at different speeds; therefore, by applying different rotor rotation speeds of the ultracentrifuge (and accordingly increasing the sedimentation factor, quantitatively measured in units of gravitational acceleration — g), fractionation of the tissue homogenate into separate subcellular structures is achieved — Table 8.1.

Class="center">Table 8.1. Subcellular structures isolated by fractionation of tissue homogenates using differential centrifugation