Tissue digestion, proteolytic

Aq solns are capable of digesting necrotic tissue, but do not attack viable tissue. Optimum proteolytic activity is at pH 5.2 to 5.5 and also at pH 7.3. Inactivated at temps above 80. ... 

The ability to identify and quantify cyanobacterial toxins in animal and human clinical material following (suspected) intoxications or illnesses associated with contact with toxic cyanobacteria is an increasing requirement. The recoveries of anatoxin-a from animal stomach material and of microcystins from sheep rumen contents are relatively straightforward. However, the recovery of microcystin from liver and tissue samples cannot be expected to be complete without the application of proteolytic digestion and extraction procedures. This is likely because microcystins bind covalently to a cysteine residue in protein phosphatase. Unless an effective procedure is applied for the extraction of covalently bound microcystins (and nodiilarins), then a negative result in analysis cannot be taken to indicate the absence of toxins in clinical specimens. Furthermore, any positive result may be an underestimate of the true amount of microcystin in the material and would only represent free toxin, not bound to the protein phosphatases. Optimized procedures for the extraction of bound microcystins and nodiilarins from organ and tissue samples are needed. 

It is possible to restore antigenicity in tissue sections by digesting the rehydrated sections with any of several proteolytic enzymes, or by incubating the tissue section at elevated temperatures in any of several liquids, including water and various buffers (antigen retrieval).12... 

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. 

It is difficult to use proteolytic enzyme digestion with this treatment, but if needed, the sections should be digested following the microwave heating because boiling enzymatically digested tissue results in tissue loss. A milder form of this method is to boil the citric acid buffer only and incubate the sections in the preheated solution, or steam, for 15 min, or sections can be heated to 80°C in this buffer and kept overnight with similar results (11). 

The combination of proteolytic enzyme digestion and heat-based treatments has been reported however, this increases the susceptibility of the tissues for disintegration. Significant shortening of the digestion time and reduction of the enzyme concentration should be undertaken when testing such protocols. Furthermore, reproducible proteolytic enzyme treatments require optimal conditions for individual tissues, which make these protocols difficult to perform. 

The role of the fibrinolytic system is to dissolve any clots that are formed within the intact vascular system and so restrict clot formation to the site of injury. The digestion of the fibrin and hence its lysis is catalysed by the proteolytic enzyme, plasmin, another serine proteinase. Plasmin is formed from the inactive precursor, plasminogen, by the activity of yet other proteolytic enzymes, urokinase, streptokinase and tissue plasminogen activator (tPA) which are also serine proteinases. These enzymes only hydrolyse plasminogen that is bound to the fibrin. Any plasmin that escapes into the general circulation is inactivated by binding to a serpin (Box 17.2). 

To prevent self-digestion, the pancreas releases most proteolytic enzymes into the duodenum in an inactive form as proenzymes (zymogens). Additional protection from the effects of premature activation of pancreatic proteinases is provided by proteinase inhibitors in the pancreatic tissue, which inactivate active enzymes by complex formation (right). 

Many proteins are formed as inactive precursors and become activated by proteolysis. The inactive precursors are termed proenzymes, zymogens or - for hormones like e.g. insulin - prehormones. Processing to the active form occius in a cell- and tissue-specific way and usually requires a specific protease. Activation can also occur intramolecu-larly by autoproteolysis. In most cases, short sequences of the protease substrate serve as a recognition signal for the attack of the processing protease. Of the numerous examples of proteolytic processing of proteases only the digestive proteases will be discussed in more detail. 

Fibrinolysis refers to the process of fibrin digestion by the fibrin-specific protease, plasmin. The fibrinolytic system is similar to the coagulation system in that the precursor form of the serine protease plasmin circulates in an inactive form as plasminogen. In response to injury, endothelial cells synthesize and release tissue plasminogen activator (t-PA), which converts plasminogen to plasmin (Figure 34-3). Plasmin remodels the thrombus and limits its extension by proteolytic digestion of fibrin. 

Semisolid samples such as muscle and liver tissues can be homogenized by blending with water or an appropriate aqueous solution such as a buffer in a mechanical or an ultrasonic device to expose the residue to the extraction solvent. Fatty tissue samples are sometimes subjected to heating at 40 or 60 C until fat becomes liquid, prior to extraction of the analytes with hexane (433) or acetonitrile (434), respectively. An alternative pretreatment approach is the enzymatic digestion of the tissue by means of proteolytic enzymes such as subtilisin A (429, 435-437). 

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