Homogenization and Subcellular Fractionation

Comporti, M., Saccocci, C. and Dianzani, M.U. (1965). EfiFect of carbon tetrachloride in vitro and in vivo on lipid peroxidation of rat liver homogenates and subcellular fractions. Enzymologja 29, 185-204. 

Lang, J.K., Gohil, K. and Packer, L. (1986) Simultaneous determination of tocopherols, ubiquinols, and ubiquinones in blood, plasma, tissue homogenates, and subcellular fractions, Anal. Biochem. 157, 106-116. 

Ojingwa, J.C. Spahn-Langguth, H. Benet, L,Z, Irreversible Binding of Tolmetin to Macromolecules via Its Glucuronide Binding to Blood Constituents, Tissue Homogenates and Subcellular Fractions In Vitro, Xenobiotica 24,495-506 (1994). 

Rosenthal HL, Cochran OA. 1972. Binding of 85Sr to homogenate and subcellular fractions of rabbit tissues. Proc Soc Exp Biol Med 141(3) 850-856. 

Our experiments have progressed stepwise from the whole animal, through tissue slices, homogenates, and subcellular fractions, to pure enzyme systems by means of which the effect of tocopherol and similar compounds can now be followed on the molecular level. Collaborators in these studies were Drs. S. Chernick, W. Mertz, M. Green, L. Corwin, Miss J. Stesney, and Mr. C. Lee. In the following, the main aspects of this work will be summarized. For experimental details and more thorough discussions the references should be consulted. 

Fig, 6. Synthesis of taurocholin and glycooholic acids in human liver homogenates and subcellular fractions obtained from patients with normal liver function, patients with obstructive jaundice, and patients with acute infectious hepatitis. 

Myelin and subcellular fractions were prepared from adult whole brain homogenates as described by Adams et al. (1963). 

The localization of a protein antigen is studied biochemically by subcellular fractionation of cell homogenates (9). Cell lysis is carried out under conditions that do not disrupt the nuclear and organelle membranes (see Protocol 3), and subcellular fractionation is achieved by centrifugation techniques such as ... 

Data are expressed as percent of homogenate activity per gram liver recovered in each fraction. Recovery is total percent of homogenate activity recovered in the various subcellular fractions. Assays were carried out at pH 6.5 and 38°. Animals had been fasted for 12 hr before use. 

The metabolic study, considered separately, consists of treatment of the animal with the radiolabeled compound followed by chemical analysis of all metabolites formed in vivo and excreted via the lungs, kidneys, or bile. Although reactive intermediates are unlikely to be isolated, the chemical structure of the end products may provide vital clues to the nature of the intermediates involved in their formation. The use of tissue homogenates, subcellular fractions, and purified enzymes may serve to clarify events occurring during metabolic sequences leading to the end products. 

Subcellular fractionation is the breaking open of a cell (e.g. by homogenization) and the separation of the various organelles from one another by centrifugation. 

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. 

Nakamura, Y., Iga, K., Shibata, T., Shudo, M., and Kataoka, K. (1993). Glial plasmalemmal vesicles A subcellular fraction from rat hippocampal homogenate distinct from synaptosomes. Glia 9, 48-56. 

Since oxidative stress has been reported to produce subcellular redistribution and activation of NF-kB, we measured total NF-kB content in cytosolic and particulate fractions of the hearts subjected to ischemia/reperfusion. In another set of experiments, the activation of NF-kB protein was studied by measuring the total NF-kB and phospho-NF-KB in the heart homogenate. Elevated ratios of NF-kB in particulate versus cytosolic fraction and of phospho-NF-KB and total NF-kB in the hearts subjected to ischemia/reperfusion were reduced by selenium. As oxidative stress has been reported to produce subcellular redistribution and activation of NF-kB and antioxidants have been shown to prevent this alteration (Cargnoni et al. 2002), it is possible that our observation on selenium-induced NF-kB translocation might be due to its antioxidant action. It should be noted that since selenium treatment could reduce the tumor necrosis factor-cc (TNF-ct)-mediated activation of NF-kB (Kim and Stadtman 1997), it suggests a possible involvement of a reduction in the formation of TNF-tx in the hearts subjected to ischemia/reperfusion by selenium. 

The disruption of tissue culture cells and their subsequent subcellular fractionation may be more difficult than parallel processes with say rat liver. Although the cells are homogeneous they are usually small and cells grown in suspension frequently have very little cytoplasm. 

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