Biological Chemistry - Berezov, T. T., Korovkin, B. F. 1998

Enzymes
Intracellular Localization of Enzymes

The question of enzyme localization within structural Cell components (Nucleus, Mitochondria, Lysosomes, etc.) is of paramount importance, particularly in preparative enzymology when researchers aim to isolate and purify an enzyme. The localization of an enzyme can be determined relatively easily using cyto- and histochemical Methods. To this end, thin tissue sections are incubated with appropriate substrates, after which the reaction product is visualized by adding suitable Reagents to produce a specific coloration.

In preparative enzymology, the method of differential centrifugation of tissue homogenates is more commonly employed (Fig. 4.26). This involves first disrupting the cellular Structure using a suitable disintegrator, and then subjecting the resulting quasi-homogeneous (homogenized) suspension to differential centrifugation at 0–4°C. Enzyme distribution is typically studied across sequential individual fractions isolated through fractional centrifugation of homogenates. These include the nuclear fraction obtained at low centrifugation speeds, the mitochondrial fraction sedimented at medium speeds, the microsomal (or ribosomal) fraction requiring high centrifugation speeds, and finally the remaining clear supernatant, which represents the soluble cytoplasmic fraction. It should be noted that the mitochondrial fraction is not homogeneous, as it also contains particles known as lysosomes, whose size is intermediate between that of mitochondria and microsomes. Likewise, the microsomal fraction is heterogeneous, consisting primarily of structurally diverse elements of The Endoplasmic reticulum.

Fractionation studies of Organ and tissue homogenates in centrifuges have demonstrated that the nuclear fraction of The Liver and Kidneys contains a relatively small number of Enzymes, even though nuclear Protein Synthesis is known to take place. As established today, the primary site of protein synthesis is the cytoplasmic ribosomal fraction. Furthermore, glycolytic enzymes have been shown to concentrate predominantly in the soluble cytoplasmic fraction, whereas cytochrome oxidase and the enzymes of The Citric Acid Cycle are localized within the mitochondrial fraction. Mitochondria are also associated with enzymes that catalyze Oxidative Phosphorylation AND fatty acid degradation. Conversely, enzymes responsible for FATTY ACID Biosynthesis are found in the soluble cytoplasmic fraction.

* For 1 atom of inorganic iron—which also catalyzes The breakdown of H₂O₂—to split the same number of H₂O₂ molecules that catalase cleaves in 1 s, it would take several years. This example clearly illustrates one of the Fundamental properties of enzymes: their exceptionally high catalytic activity.

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Fig. 4.26. Differential centrifugation of liver tissue homogenates (diagram); g represents the relative centrifugal force.

The Isolation and Purification of enzymes from biological sources in a pure (homogeneous) state utilize the full arsenal of methods available for isolating individual Proteins (see Chapter 1).