Purification subcellular fractionation

The protein has to be obtained in solution prior to its purification. Thus tissues and cells must be disrupted by homogenization or osmotic lysis and then subjected to differential centrifugation to isolate the subcellular fraction in which the protein is located. For membrane-bound proteins, the membrane structure has to be solubilized with a detergent to liberate the protein. 

Work continues in two areas purification of the lytic system to allow protease and glucanase levels to be controlled independently and investigation of the release of site-specific yeast enzymes and subcellular fractions by enzymatic lysis. 

Separation and Purification A high degree of purification of the homogenized subcellular fractionations into individual proteins (rarely achieved even by two-dimensional-PAGE for all proteins) or a simple mixture is an essential requirement for unambiguous protein identification. This aspect was also discussed in Section 8.3. The use of several orthogonal techniques (e.g., 2-DE or LC) becomes essential to reduce the complexity of the sample and improve chances of detection of low-abundance proteins. 

In vitro methods are performed outside the whole organism. They are essentially test tube methods, employing crude cell homogenates, subcel-lular fractions thereof, or purified enzymes. For methods of cell disruption, subcellular fractionation and enzyme purification, see Proteins Density gradient centrifugation. 

Admittedly, the data from aorta are rather skimpy. Further progress waits upon adequate volumes of subcellular fractions so that purification procedures can be utilized. Access to mammals with large aortas and technical innovations permitting dissolution of the tough connective tissue so that subcellular fractions can be obtained without trauma are methodologic advances desperately needed to allow modem biochemical approaches to aortic metabolism. 

Aarsman AJ, de Jong JGN, Arnoldussen E, Neijs FW, van Wassenaar PD, Van den Bosch H. Immunoaffinity purification, partial sequence and subcellular fractionation of rat liver phospholipase A2. J Biol Chem 1989 264 10008-10014. 

The enzyme was originally found to be membrane bound and resisted solubilization and purification (26). Lundblad and Moore (27), however, have reported solubilizing it using dilute (5 mM) sodium borate buffer at pH 9 after 16 hr at 37°. Studies on regional and subcellular distribution using density gradient techniques have revealed that the 2, 3 -cyclic phosphate diesterase concentrates in those fractions containing myelin (28, 29), and the conclusion has been reached that the enzyme is localized in the myelin sheath or intimately associated structures. Kurihara and... 

Figure 5.1 Anatomy of a purification scheme the starting and ending points for three types of purification. (/) The starting samples are composed of cells and exracellular materials, the goal of the purification is their separation. (//) The source of the enzyme is a heterogeneous population of cells, and the goal is to produce separate homogeneous populations (1-5). (Ill) The source of the enzyme is a cell, and the goal is to isolate a subcellular organelle, fraction, or purified protein.

In general, the aim of subcellular analysis is to quantify an analyte within a specific subcellular compartment. Consequently, in most cases, an organelle fraction must be purified before analysis. Cellular fractionation, that is, isolation and purification of organelles, has been indispensable in the biochemical fields and, as evidenced in the literature, has been used pervasively. Since complete reviews can be found in the biochemical literature, we will only briefly describe the principles of cellular fractionation. 

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