Organelle separations

The first step is not required for chemically synthesized products, otherwise prior cell disruption and organelle separation are required to yield cell-free extract from which the desired biomacromolecule can be purified from its natural enviromnent. Total cellular or tissue proteins may be solubilized and assayed prior to purification (Shaw, 1998). Different approaches are available to lyse cells (http //expasy.cbr.nrc.ca/ch2d/protocols/). An approach can be as gentle as adding a surfactant or subjection to an osmotic shock, or can be more energetic such as ultra-sonification, bead beater or French press. Table 3.1 lists some of common methods for cell disruption. 

Organelles separated from cytoplasm by a double membrane are the nucleus, mitochondria, and plastids. Secondary products and enzymes of secondary metabolism are rare in nuclei and mitochondria, but common in plastids. The envelope and the invaginations of the inner membrane, which traverse these organelles, and the nonparticulate fraction obtained after chloroplast rupture... 

Embryos were obtained from rape plants as previously described. The embryos were homogenized and membranes and organelles separated and fractionated using sucrose density gradient centrifugation... 

The DEP of numerous particle types has been studied, and many apphcations have been developed. Particles studied have included aerosols, glass, minerals, polymer molecules, hving cells, and cell organelles. Apphcations developed include filtration, orientation, sorting or separation, characterization, and levitation and materials handhng. Effects of DEP are easily exhibited, especially by large particles, and can be apphed in many useful and desirable ways. DEP effects can, however, be observed on particles ranging in size even down to the molecular level in special cases. Since thermal effects tend to disrupt DEP with molecular-sized particles, they can be controlled only under special conditions such as in molecular beams. 

Intracellular motility is also of vital importance in the lives of cells and the organisms they form. Material and organelles are transported within cells along microtubules and microfilaments an extreme example of this are the axons of nerve cells which transport materials to the synapses where they are secreted—another motile event. Other examples of intracellular motility include phagocytosis, pino-cytosis, the separating of chromosomes and cells in cell division, and maintenance of cell polarity. 

Compartmentation of pathways in separate subcellular compartments or organelles permits integration and regulation of metabolism. Not all pathways are of equal importance in all cells. Figure 15-7 depicts the subceh lular compartmentation of metabohc pathways in a hepatic parenchymal cell. 

Because of the difficulties in abundance and compatibility described above, fractionation steps are often performed on protein mixtures prior to 2D gel separation to reduce the complexity of the mixtures. Prefractionation of proteins can be achieved by (i) isolation of cell compartments such as the plasma membrane or organelles such as mitochondria or nuclei, (ii) by... 

Figure 2.2. Fractionation of protein extracts before 2D gel electrophoresis. Crude lysates can be fractionated by affinity purification or by a number of chromatographic techniques. In addition, organelles or other cellular structures can be purified and lysates from these organelles can be fractionated or separated directly on 2D gels. By repeating this procedure using a number of conditions it may be possible to visualize a large fraction of a cell s proteome.

Bacteria, being procaryotic, do not show compartmentation of the biosynthetic processes. The genome of a bacterium relates directly to the cytoplasm of the cell. Transcription into mRNA can lead directly to translation, and the processes of transcription and translation are not carried out in separate organelles. Animal cells, being eucaryotic, show compartmentation of the transcription and translation processes. Transcription of the genome into mRNA occurs in the nucleus, whereas translation occurs in the cytoplasm. The messenger RNA in the eucaryote is usually modified by adding to it... 

The information contained in the DNA (i.e., the order of the nucleotides) is first transcribed into RNA. The messenger RNA thus formed interacts with the amino-acid-charged tRNA molecules at specific cell organelles, the ribosomes. The loading of the tRNA with the necessary amino acids is carried out with the help of aminoacyl-tRNA synthetases (see Sect. 5.3.2). Each separate amino acid has its own tRNA species, i.e., there must be at least 20 different tRNA molecules in the cells. The tRNAs contain a nucleotide triplet (the anticodon), which interacts with the codon of the mRNA in a Watson-Crick manner. It is clear from the genetic code that the different amino acids have different numbers of codons thus, serine, leucine and arginine each have 6 codewords, while methionine and tryptophan are defined by only one single nucleotide triplet. 

Finally, note that eukaryote chemotypes have as a general feature the increase inside the cell vesicles of elements, here calcium, previously confined to the outside of prokaryotes, but these increases are different in different vesicles and organelles and in different organisms separating cells into different chemotypes. This is also seen in their minerals. There is as yet far too little quantitative analysis of calcium or indeed of elements generally to allow us to build a full picture of chemotypes together with genotypes (see Table 8.22). 

Separate management of radical photo- and oxygen chemistry in organelles... 

If the allelochemical is hydrophylic, it cannot enter into the cell and act from outside by binding with chemoreceptors. The compounds from allelopathically active plants may serve as chemosignals and their signalling occurs via alternative pathways (i) Chemoreceptor (sensors) — transducers (G-proteins) —> secondary messengers (Ca2+, cyclic AMP or GMP, inositol triphospate, etc) —> organelles or (ii) Chemoreceptor (sensors) —> ion channels —> action potential organelles, or (iii) Chemoreceptor (sensors) —> ion channels —> cytoskeleton— organelles (Roshchina, 2005 a). What is the effect of acted allelochemical on the pathways, could be analysed to study the effects of substances on separate sites of the transduction chain. 

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