Fractionation of cells and tissues

Fractionation of Cells and Tissues 139. Scalar coupling ( coupling)... 

The Human Proteome Project (HUPO, www.hupo.org) initiated the Plasma Proteome Project (PPP) in 2002, and numerous laboratories have contributed to this ambitious project of deciphering all proteins contained in the human plasma. Plasma, the soluble component of the human blood, is believed to harbor thousands of distinct proteins that originate from a variety of cells and tissues through either active secretion or leakage from blood cells or tissues. For the reasons described above, HUPO recommends use of plasma instead of serum, with EDTA (or citrate) for anticoagulation and standardized sample preparation. HUPO proposes combinations of serum depletion, fractionation procedures, and MS/MS technologies, with explicit... 

The metabolic pathways for BaP are summarized in Fig. 6. Many of these reactions have been elucidated in studies with rat liver microsomes although investigations with whole cells or in organ culture have also contributed, especially in the case of conjugates. Studies on BaP metabolism in vitro are summarized in Table X. Table X is not an exhaustive review of the literature, but rather is intended to present a cross-section of studies performed with subcellular fractions, whole cells, and tissues in organ culture. We have emphasized studies in which particular metabolites were identified or assayed, usually by thin-layer... 

A detailed discussion of the modes of occurrence and biological importance of the polynucleotides is outside the scope of this article. However, in examining the structures of polynucleotides, it is necessary to take into consideration the origins of the materials studied. The pioneer researches of Caspersson114 indicated that deoxyribonucleic acids are present exclusively in the nucleus, whereas ribonucleic acids are found chiefly in the cytoplasm and only to a small extent in the nucleus. This general outline of the distribution of nucleic acids within the cell has been confirmed and extended by more recent work,116 and it has been possible to isolate both types of nucleic acid from different cellular fractions of the same tissue.116... 

The FAS multi-enzyme complex synthesizes saturated C16 fatty acids, but cells and tissues need unsaturated and longer chain fatty acids. The palmitoyl-CoA can be modified by either chain elongation and/or oxidation in order to produce different fatty acid molecules. Both elongation and desaturation occur within the smooth endoplasmic reticulum (SER, microsomal fraction) of the cell. 

In exploring the biological role of lipids in cells and tissues, it is essential to know which lipids are present and in what proportions. Because lipids are insoluble in water, their extraction and subsequent fractionation require the use of organic solvents and some techniques... 

The next step is to select a protocol for isolating cell walls that suits the type of material to be investigated and the reasons for doing the research (see Critical Parameters). Two basic protocols are described, one for plant tissue that does not contain starch (see Basic Protocol 2) and one for plant tissue that does contain starch (see Basic Protocol 3), as well as three alternate protocols that can be used and modified to suit (see Commentary). The final step is fractionation of cell wall polysaccharides, which is a sequential chemical extraction of polysaccharides from the walls (see Basic Protocol 4). Table E3.1.1 provides a more detailed description of the protocols presented in this unit. 

Protein expression of dysbindin-1 is also ubiquitous in the body and is detectable in cell bodies of virtually all neuronal populations. Levels of somatal protein are variable, however, with the highest levels found in areas listed above where gene expression is highest. High levels of dysbindin-1 protein expression are also seen in certain synaptic fields. Where these have been examined with immunoEM, dysbindin-1 has been found mainly along microtubules of dendrites and axons, in PSDs of dendritic spines, and around synaptic vesicles. Tissue fractionation of whole brain tissue reveals that dysbindin-1 A is most highly concentrated in PSD fractions, dysbindin -IB in synaptic vesicle fractions, and dysbindin-1C in both PSD and synaptic vesicle fractions. 

A useful procedure for estimating adenylate cyclase in intact cells and tissues is to incubate the tissue with labelled adenine and then measure the rate of labelling of cyclic AMP [112]. Adenine readily penetrates cells and is partially converted to ATP. In heart slices, the ATP newly synthesised from radioactive adenine was found in equilibrium with the existing pool used for the production of cyclic AMP the specific activity of the newly-formed cyclic AMP was similar in the presence and in the absence of stimulatory hormone [113]. The prelabelling method has been compared with the protein-binding method in brain slices [114,115]. Increases in total levels of cyclic AMP and increases in levels of radioactive cyclic AMP derived from intracellular adenine nucleotides labelled by prior incubation with radioactive adenine occurred on similar time courses and to similar extents. Radioactive cyclic AMP represented a small (7-13%) but relatively constant fraction of the total amount of cyclic AMP. These results provided no evidence for the presence of more than one major compartment of adenine nucleotides in brain slices that serve as a source of nucleotide precursor for cyclic AMP. The nucleotides of this compartment were uniformly labelled by incubation with radioactive adenine [116]. 

A8. Anderson, L., and Anderson, N. G., Analytical techniques lor cell fractions. XXII. Two-dimensional analysis of serum and tissue proteins Multiple gradient-slab gel electrophoresis. Anal. Biochem. 85, 341-354 (1978). 

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