Isolation and Subfractionation

Among the more compelling evidences in support of the reality of the Golgi apparatus was the demonstration in 1953 by Schneider and coworkers (Schneider et al., 1953; Schneider and Kuff, 1954; Dalton and Felix, 1954; Kuff and Dalton, 1959) of recognizable Golgi apparatus material from epididymal homogenates by a density gradient technique with light microscopy to evaluate the procedure. Cells were disrupted in a sucrose medium containing 0.34 M (2%) sodium chloride using a loose-fitting homogenizer. Golgi apparatus ranging in size from 0.5 μ upward (average 1.2 μ) had isopycnic densities between 1.09 and 1.13. The possibility of contamination was recognized. In a subsequent study (Kuff and Dalton, 1959), the procedure was modified to include flotation of the membrane material to its equilibrium position and the product was examined by electron microscopy. Unfortunately, these preparations were insufficiently characterized to provide new information on Golgi apparatus structure and function but they paved the way for future efforts in 1964 with the isolation of Golgi apparatus from plant cells (Morré and Mollenhauer, 1964) and culminating in 1968 with the isolation of intact Golgi apparatus from rodent livers in sufficient yield and fraction purity to permit definitive biochemical studies (Morré et al., 1968a, b).

Categorically, the Golgi apparatus was the last major cell component to be isolated from mammalian cells. A factor was the lack of a unique biochemical function or property that could be ascribed to the cell component to provide an independent biochemical marker to aid in its isolation. As detailed in Chapter 2, the definition of Golgi apparatus was, and continues to be, based on morphology. Therefore, it is logical that the basis for their initial isolation, however tedious, was by necessity based on morphology (Hamilton et al., 1967; Ovtracht et al., 1969; Morré et al., 1970a). A summary of the development of Golgi apparatus isolation methods and the many contributions from all different laboratories is given in Chapter 7 as they relate to biochemical studies of the Golgi apparatus.


Golgi Apparatus Secretory Vesicle Fraction Purity Rodent Liver Sucrose Layer 
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© Springer-Verlag Berlin Heidelberg 2009

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