Eukaryotic Cells Have a Variety of Membranous Organelles, Which Can Be Isolated for Study

Typical eukaryotic cells (Fig. 1–1) are much larger than prokaryotic cells—commonly 5 to 100 μm in diameter, with cell volumes a thousand to a million times larger than those of bacteria. The distinguishing characteristics of eukaryotes are the nucleus and a variety of membranebounded organelles with specific functions: mitochondria, endoplasmic reticulum, Golgi complexes, and lysosomes.  Plant cells also contain vacuoles and chloroplasts (Fig. 1–1). Also present in the cytoplasm of many cells are granules or droplets containing stored nutrients such as starch and fat. In a major advance in biochemistry,   Albert Claude, Christian de Duve, and George Palade developed methods for separating organelles from the cytosol and from each other—an essential step in isolating biomolecules and larger cell components and investigating their structures and functions. In a typical cell fractionation  (Fig. 1–2), cells or tissues in solution are disrupted by gentle homogenization. This treatment ruptures the plasma membrane but leaves most of the organelles intact.

FIGURE 1–1 Eukaryotic cell structure. Schematic illustrations of the two major types of eukaryotic cell: (a) a representative animal cell and (b) a representative plant cell. Plant cells are usually 10 to 100 μm in diameter—larger than animal cells, which typically range from 5 to 30 μm. Structures labeled in red are unique to either animal or plant cells.

The homogenate is then centrifuged; organelles such as nuclei, mitochondria, and lysosomes differ in size and therefore sediment at different rates. They also differ in specific gravity, and they “float” at different levels in a density gradient.

  Differential centrifugation results in a rough fractionation of the cytoplasmic contents, which may be further purified by isopycnic (“same density”) centrifugation. In this procedure, organelles of different buoyant densities  (the result of different ratios of lipid and protein in each type of organelle) are separated on a density gradient. By carefully removing material from each region of the gradient and observing it with a microscope, the biochemist can establish the sedimentation position of each organelle and obtain purified organelles for further study.

FIGURE 1–2 Subcellular fractionation of tissue. A tissue such as liver is first mechanically homogenized to break cells and disperse their contents in an aqueous buffer. The sucrose medium has an osmotic pressure similar to that in organelles, thus preventing diffusion of water into the organelles, which would swell and burst. (a) The large and small particles in the suspension can be separated by centrifugation at different speeds, or (b) particles of different density can be separated by isopycnic centrifugation. In isopycnic centrifugation, a centrifuge tube is filled with a solution, the density of which increases from top to bottom; a solute such as sucrose is dissolved at different concentrations to produce the density gradient. When a mixture of organelles is layered on top of the density gradient and the tube is centrifuged at high speed, individual organelles sediment until their buoyant density exactly matches that in the gradient. Each layer can be collected separately.

  For example, these methods were used to establish that lysosomes contain degradative enzymes, mitochondria contain oxidative enzymes, and chloroplasts contain photosynthetic pigments. The isolation of an organelle enriched in a certain enzyme is often the first step in the purification of that enzyme.