Subcellular Organelles

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▶Nuclei: The eukaryotic nucleus carries the genetic information of the cell in multiple chromosomes, each containing a single DNA molecule.The nucleus is bounded by a lipid double membrane, the nuclear envelope, containing pores which allow passage of moderately large molecules. Transcription of RNA takes place in the nucleus and the processed RNA molecules pass into the cytoplasm where translation takes place. Nucleoli are bodies within the nucleus where rRNA is synthesized and ribosomes are partially assembled

▶Mitochondria and chloroplasts: Cellular respiration, that is the oxidation of nutrients to generate energy in the form of adenosine 5′-triphosphate (ATP), takes place in the mitochondria. These organelles are roughly 1–2 μm in diameter and there may be 1000–2000 per cell. They have a smooth outer membrane and a convoluted inner membrane that forms protrusions called cristae. They contain a small circular DNA molecule, mitochondrial-specific RNA and ribosomes on which some mitochondrial proteins are synthesized. However, the majority of mitochondrial (and chloroplast) proteins are encoded by nuclear DNA and synthesized in the cytoplasm. These latter proteins have specific signal sequences that target them to the mitochondria. The chloroplasts of plants are the site of photosynthesis, the light-dependent assimilation of CO2 and water to form carbohydrates and oxygen. Though larger than mitochondria, they have a similar structure except that, in place of cristae, they have a third membrane system (the thylakoids) in the inner membrane space. These contain chlorophyll, which traps the light energy for photosynthesis. Chloroplasts are also partly genetically independent of the nucleus. Both mitochondria and chloroplasts are believed to have evolved from prokaryotes which had formed a symbiotic relationship with a primitive nucleated eukaryote.

▶Endoplasmic reticulum: The endoplasmic reticulum is an extensive membrane system within the cytoplasm and is continuous with the nuclear envelope. Two forms are visible in most cells. The smooth endoplasmic reticulum carries many membrane-bound enzymes, including those involved in the biosynthesis of certain lipids and the oxidation and detoxification of foreign compounds (xenobiotics) such as drugs. The rough endoplasmic reticulum (RER) is so-called because of the presence of many ribosomes. These ribosomes specifically synthesize proteins intended for secretion by the cell, such as plasma or milk proteins, or those destined for the plasma membrane or certain organelles. Apart from the plasma membrane proteins, which are initially incorporated into the RER membrane, these proteins are translocated into the interior space (lumen) of the RER where they are modified, often by glycosylation. The lipids and proteins synthesized on the RER are transported in specialized transport vesicles to the Golgi complex, a stack of flattened membrane vesicles which further modifies, sorts and directs them to their final destinations.

▶Microbodies: Lysosomes are small membrane-bound organelles which bud off from the Golgi complex and which contain a variety of digestive enzymes capable of degrading proteins, nucleic acids, lipids and carbohydrates. They act as recycling centers for macromolecules brought in from outside the cell or from damaged organelles. Some metabolic reactions which generate highly reactive free radicals and hydrogen peroxide are confined within organelles called peroxisomes to prevent these species from damaging cellular components. Peroxisomes contain the enzyme catalase, which destroys hydrogen peroxide:

2H2O2 –> 2H2O + O2

Glyoxysomes are specialized plant peroxisomes which carry out the reactions of the glyoxylate cycle. Lysosomes, peroxisomes and glyoxysomes are collectively known as microbodies.

▶Organelle isolation: The plasma membrane of eukaryotes can be disrupted by various means including osmotic shock, controlled mechanical shear or by certain non ionic detergents. Organelles displaying large size and density differences, for example nuclei and mitochondria, can be separated from each other and from other organelles by differential centrifugation according to the value of their sedimentation coefficients. The cell lysate is centrifuged at a speed which is high enough to sediment only the heaviest organelles, usually the nuclei. The supernatant containing all the other organelles is removed then centrifuged at a higher speed to sediment the mitochondria, and so on. This technique is also used to fractionate suspensions containing cell types of different sizes, for example red cells, white cells and platelets in blood. These crude preparations of cells, nuclei and mitochondria usually require further purification by density gradient centrifugation. This is also used to separate organelles of similar densities.

In rate zonal centrifugation, the mixture is layered on top of a pre-formed concentration (and, therefore, density) gradient of a suitable medium in a centrifuge tube. Upon centrifugation, bands or zones of the different components sediment at different rates depending on their sedimentation coefficients, and separate. The purpose of the density gradient of the supporting medium is to prevent convective mixing of the components after separation (i.e. to provide stability) and to ensure linear sedimentation rates of the components (it compensates for the acceleration of the components as they move further down the tube).

In equilibrium (isopycnic) centrifugation, the density gradient extends to a density higher than that of one or more components of the mixture so that these components come to equilibrium at a point equal to their own density and stop moving. In this case, the density gradient can either be pre-formed, and the sample layered on top, or self-forming, in which case the sample may be mixed with the gradient material. Density gradients are made from substances such as sucrose, Ficoll (a synthetic polysaccharide), metrizamide (a synthetic iodinated heavy compound) or cesium chloride (CsCl), for separation of nucleic acids. Purity of the subcellular fraction can be determined using an electron microscope or by assaying enzyme activities known to be associated specifically with particular organelles, for example succinate dehydrogenase in mitochondria.