Cell disruption osmotic lysis

The subcellular location of PG was studied in cells disrupted by osmotic lysis through formation and disruption of sphaeroplasts from self-induced anaerobically-grown cells. A discontinuous sucrose-density gradient produced four bands labelled I, II, III and IV. Band I included many vesicles and a peak of alkaline phosphatase activity (a vacuolar marker in yeasts), NADPH cytochrome c oxidoreductase activity, an endoplasmic reticulum marker, and... 

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. 

Osmotic lysis The plasma membranes of cells are water-permeable but are impermeable to large molecules and some ions. Thus, if cells are placed into water or dilute buffer, they swell owing to the osmotically driven influx of water. Since the plasma membrane is not able to stretch very much (the red blood cell membrane can stretch only up to 15 percent of its normal area before disruption), the cells burst. The method is effective for isolated cells but is not so effective for tissues. 

In addition to these various chemical treatment methods, a number of physical methods of cell disruption can be used in a chip-based system. These physical methods include osmotic shock, which occurs when cells are suspended in a hypotonic solution shearing and fracturing of cells walls and membranes using microfabricated needles or spherical particles (beads) application of an electric field that causes electroporation ultrasonication of the cell sample and thermal lysis. 

Electrophiles attack both the cell wall and the macromolecules within a cell, such as proteins, hpids, carbohydrates, and nucleic acids. The oxidizers exhibit rapid kill, while moderate electrophiles react more slowly to enter into a cell to disrupt the internal macromolecules. (Note the oxidizing biocides can also react with the EPS or slime to create a reaction barrier to limit entry of the biocide into the cell itself) Membrane active biocides disrupt the structure and function of the cell membrane. This leads to osmotic lysis and leaking of intracellular material. The protonophores are generally not used for water treatment because their rate of kill is slow and they are ineffective against gram-negative bacteria. ... 

The isolation of bacterial DNA described in this experiment, patterned after the work of Marmur (1961), accomplishes these objectives. Bacterial cells are disrupted by initial treatment with the enzyme, egg-white lysozyme, which hydrolyzes the peptidoglycan that makes up the structural skeleton of the bacterial cell wall. The resultant cell walls are unable to withstand osmotic shock. Thus, the bacteria lyse in the hypotonic environment. The detergent, sodium dodecyl sulfate, (SDS, sodium do-decyl sulfate) then completes lysis by disrupting residual bacterial membranes. SDS also reduces harmful enzymatic activities (nucleases) by its ability to denature proteins. The chelating agents, citrate and EDTA (ethylenediamine tetraacetic acid), also inhibit nucleases by removing divalent cations required for nuclease activity. 

Cell lysis or autolysis Osmotic disruption of cell membrane under high osmotic pressure (usually 20% sucrose) or detergent. Autolysis (e.g. yeast) is generally carried out in the presence of toluene. 

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