Osmotic lysis

Several drugs in current medical use are mechanism-based enzyme inactivators. Eor example, the antibiotic penicillin exerts its effects by covalently reacting with an essential serine residue in the active site of glycoprotein peptidase, an enzyme that acts to cross-link the peptidoglycan chains during synthesis of bacterial cell walls (Eigure 14.17). Once cell wall synthesis is blocked, the bacterial cells are very susceptible to rupture by osmotic lysis, and bacterial growth is halted. 

Penicillin has an interesting mode of action it prevents the cross-linking of small peptide chains in peptidoglycan, the main cell wall polymer of bacteria. Pre-existing cells are unaffected, but all newly produced cells are abnormally grown. The newborn cells are unable to maintain their wall rigidity, and they are susceptible to osmotic lysis. 

The spherocytes are much more susceptible to osmotic lysis than are normal red blood cells. This is assessed in the osmotic fragility test, in which red blood cells are exposed in vitro to decreasing concentrations of NaCl. The physiologic concentration of NaCl is... 

Sphaeroplasts were prepared by slight modifications to published methods [12,13]. Lysis of sphaeroplasts was effected by a combination of osmotic lysis and gentle mechanical disruption [14]. Discontinuous sucrose-density gradients were constructed and fractions were then assayed for protein, PG and marker enzymes for different organelles. 

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... 

Figure 1.14. Complement activation via the classical pathway. The sequential activation of complement following antibody deposition onto a surface is shown. C9 forms a pore in the membrane, eventually leading to cell death by osmotic lysis. See text for details.

Njogu, R.M. Kiaira, J.K Trypanosoma brucei a quick method for separating blood-stream trypomastigotes from infected blood by differential osmotic lysis. Comp. Biochem. Physiol. A, 71, 265-269 (1982)... 

Several mechanisms have evolved to prevent this catastrophe. In bacteria and plants, the plasma membrane is surrounded by a nonexpandable cell wall of sufficient rigidity and strength to resist osmotic pressure and prevent osmotic lysis. Certain freshwater protists that live in a highly hypotonic medium have an organelle (contractile vacuole) that pumps water out of the cell. In multicellular animals, blood plasma and interstitial fluid (the extracellular fluid of tissues) are maintained at an osmolarity close to that of the cytosol. The high concentration of albumin and other proteins in blood plasma contributes to its osmolarity. Cells also actively pump out ions such as Na+ into the interstitial fluid to stay in osmotic balance with their surroundings. 

Osmosis also has consequences for laboratory protocols. Mitochondria, chloroplasts, and lysosomes, for example, are bounded by semipermeable membranes. In isolating these organelles from broken cells, biochemists must perform the fractionations in isotonic solutions (see Fig. 1-8). Buffers used in cellular fractionations commonly contain sufficient concentrations (about 0.2 m) of sucrose or some other inert solute to protect the organelles from osmotic lysis. 

Osmotic lysis consists of suspending cells in a solution of relatively high ionic strength. This causes water inside the cell to diffuse out through the membrane. The cells are then isolated by centrifugation and transferred to pure water. Water rapidly diffuses into the cell, bursting the membrane. 

To study transport in the absence of complicating metabolic processes, it often is advantageous to work with isolated membrane vesicles rather than with whole cells. Cytoplasmic membrane vesicles can be obtained from either eukaryotic or bacterial cells after homogenization or osmotic lysis. Transport proteins that have been solubilized with detergents also can be reincorporated into synthetic phospholipid vesicles (fig. 17.27). 

Cell lysis Mechanical methods pressure shearing, ultrasonic disintegration, bead-mill homogenizers Nonmechanical methods enzymatic lysis, osmotic lysis, freezing and thawing, detergent-based lysis and electroporation... 

Osmotic lysis Not required, inexpensive Gentle Osmotic shock... 

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. 

Okada, C.Y. and Rechsteiner, M. (1982) Introduction of macromolecules into cultured mammalian cells by osmotic lysis of pinocytic vesicles. Cell 29, 33-41. 

For complement-dependent cytotoxicity, a complex cascade of protein binding and cleavage occurs that culminates in final complement mediated effector functions of phagocytosis, recruitment and activation of leukocytes, and osmotic lysis. It has been reported that Rituximab (anti-CD20) utilizes a complement-dependent mechanism for tumor cell destruction [14], If the desired effect of a therapeutic antibody is complement-dependent activity, the isotype subgroup chosen should be one that binds to complement proteins (IgGl or IgG3). 

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. 

The membrane-attack complex (MAC) is the cytolytic end product of the complement cascade it forms a transmembrane channel, leading to osmotic lysis of the target cell (Fignre 15.1). 

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