Proteolytic enzymes, cell dissociation

The tissue or cell sample is firstly homogenized in a buffer containing a detergent such as Triton X-100 and sodium deodecyl sulphate (SDS), which disrupts the cell and dissociates DNA-protein complexes. Protein and RNA are then removed by sequential incubations with a proteolytic enzyme (usually proteinase K) and ribonuclease. Finally the DNA is extracted into ethanol. Ethanol only precipitates long chain nucleic acids and so leaves the single nucleotides from RNA digestion in the aqueous layer. 

Since protein complex formation and Ca2+ are critical to cell fixation within a tissue, dissociation media usually contain some type of proteolytic enzyme and the Ca2+ chelator, EDTA. The proteolytic enzyme can be of general specificity, such as trypsin, or can be a more targeted enzyme, such as a collagenase selective for the collagen-type characteristic of the tissue of interest. Hyaluronidase has been also used with matrix rich in hyaluronic acid, such as for isolation of duodenal entero-cytes. In all cases, the appropriate incubation times and concentrations to achieve cell dispersal, but retain high viability, need to be determined empirically. One factor... 

For isolating all DNA contained in a cell, the cell culture or tissue sample is transferred into a buffer which contains a detergent such as SDS or Triton A-100. The detergent disrupts the cellular walls and dissociates any DNA-protein complexes. RNA molecules contained in the cell extract are broken up by treatment with a ribonuclease. Proteins can be digested by a proteolytic enzyme, most commonly proteinase K. The DNA can then be extracted from the mixture by precipitation with ethanol. Only long nucleic acid chains precipitate, single nucleotides and products of the RNA digestion remain in solution. 

A common method for RNA isolation is the proteinase K method. In this method, the cells are lysed by incubation in a hypotonic solution followed by centrifugation to remove DNA and cell debris. Treatment with the proteolytic enzyme proteinase K leads to the dissociation of RNA-protein complexes and the digestion of the proteins. The digestion products are then removed by phenol/chloroform extraction and the RNA in the remaining aqueous solution is precipitated using ethanol. 

The hydrophilic cobalamin molecule has to be transported from the intestine to the blood by an elaborate transfer system. Cobalamin is first released from binding substances in the food by peptic activity at low pH in the stomach and becomes bound to so-called R-binders present in saliva and gastric juice. In the ileum pancreatic proteolytic enzymes dissociate the cobalamins from the R-binders and in this way facilitate their binding to Intrinsic Factor, a glycoprotein that in man is synthesized and released by the gastric parietal cells. The cobalamin-Intrinsic Factor complex becomes attached to specific receptors on the mucosal surface of the distal part of the ileum (45). Through an incompletely defined mechanism cobalamin enters the mucosal cell and is passed on to the plasma transport protein transcobalamin II (46). This 38,000-MW polypeptide carries cobalamin through the portal circulation first to the liver and distributes it from there to the other tissues. 

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