Cell organelle isolation

CTC, used extensively to monitor calcium release in both whole cells and isolated organelles (28-33), is an amphipathic molecule that easily passes through cell membranes (see Figure 1). The fluorescence of this probe is enhanced more than fiftyfold by binding of calcium when the dye is intercalated into biological membranes. 

One striking advance in modem subcellular biology is a more complete understanding of the interrelationship of certain plant cell organelles (Fig. 2A-F). Rather than existing as discrete entities in physiological and biochemical isolation of one another, it is now generally accepted... 

Organelle isolation from plant cells is an important technique for the investigation of the physiological functions of the organelle itself. Due to... 

Methods for the Identification of Isolated Plant Cell Organelles... 

Because disrupted tissue preparations were unsatisfactory, attempts were made to work either with more organized systems such as tissue slices (liver-Krebs) or to identify and isolate the intracellular organelles involved in the reactions. Cytochemical procedures were developed in the 1930s and 1940s to locate sites of reaction in situ in cells (Chapter 9). Examination of cell ultrastructure became possible when the electron microscope was introduced after 1945. Techniques for the isolation of cell organelles, notably mitochondria, were developed about this time (Chapter 9). 

In some cases it is necessary to isolate cell organelles before Vn x is measured (Figure 3.16). A number of cells, a whole tissue or a sample of a tissue is taken, the cells are broken and the organelle in which the enzyme is present is separated, usually by centrifugation. The organelle is then extracted to provide a medium which can be used to measure the enzyme activity by a specific method. 

The plasma membrane is the outermost part of the cell. It isolates the cytoplasm. It regulates what comes in and out of the cytoplasm. It also allows interaction with other cells. The cytoplasm is the second layer of the cell. It contains water, salt, enzymes, and proteins. It also contains organelles like mitochondria. 

To investigate the individual compartments of the cell (see p. 196), various procedures have been developed to enrich and isolate cell organelles. These are mainly based on the size and density of the various organelles. 

Non-invasive assessments of mitochondrial function in vivo typically reflect the techniques to monitor it in cells or isolated organelles. Direct calorimetry, organismal oxygen consumption, CO2 production and a host of other techniques all provide insight into mitochondrial capacity, albeit with confounding factors of movement and physical training of the subject. 

Lipoproteins or glycoproteins are the macromolecules that most commonly form receptors. They are often firmly embedded in the plasma membrane or cell-organelle membrane as intrinsic proteins (see section 7.1). At times, this renders their isolation and subsequent functional reconstitution difficult, as their structure may be dependent upon the surrounding membrane. Isolation of such a receptor molecule may cause its structural collapse, even to the extent that specific binding properties are lost. 

Within the living cell the great majority of the enzymes are attached to membrane structures or contained in cell organelles. When enzymes are isolated, they are removed from their natural state and quite often are highly unstable. The artificial binding of enzymes into membranes makes possible study of the interaction between diffusion and enzyme reaction within a well-defined context.1... 

In situ hybridization may be defined as the detection of nucleic acids in situ in cells, tissues, chromosomes, and isolated cell organelles. The technique was described in 1969 by two separate groups who demonstrated repetitive riboso-mal sequences in nuclei of Xenopus oocytes using radiolabeled probes (1,2). Refinements in recombinant DNA technology and the development of nonisotopic probe labeling and detection (3) obviate the need for radiation protection and disposal facilities, and have converted nonisotopic in situ hybridization (NISH) from a purely research technique to one that can be used in routine laboratory testing. 

Initially plant cell culture techniques progressed more slowly than those for animal cells, as did procedures for the isolation of plant cell organelles. Technical difficulties have now largely been overcome and culturing plant embryos has now found important commercial applications. 

A sample of rat liver was homogenized, the various cell organelles were isolated by differential centrifugation, and the succinate dehydrogenase enzymatic activity in each organelle preparation was determined ... 

Nishimura, M., Graham, D., and Akazawa, T. 1976. Isolation of intact chloroplasts and other cell organelles from spinach leaf protoplasts. Plant Physiol. 58, 309-314. 

Methods utilizing isolated, perfused whole organs and cells or cell organelles... 

The second level of decision making concerns the form of the biocatalyst, i.e., a whole cell, a cell organelle, an enzyme complex, a crude enzyme preparation, or an isolated enzyme, aU either free or immobilized. Availability, price, cofactor need, etc., are points that must be considered at this level. The costs associated with immobilization obviously should be regained by the possibility of developing a more efficient process. 

Biosensors using higher integrated biocatalytic phases, i.e. cell organelles, intact cells, and tissue material, are compared with isolated enzyme sensors. The merits of the former in the determination of complex variables , such as mutagenicity and nutrient content, are outlined. 

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