Self-digestion

In the area of a gastric or duodenal peptic ulcer, the mucosa has been attacked by digestive juices to such an extent as to expose the subjacent connective tissue layer (submucosa). This self-digestion occurs when the equilibrium between the corrosive hydrochloric acid and acid-neutralizing mucus, which forms a protective cover on the mucosal surface, is shifted in favor of hydrochloric acid. Mucosal damage can be promoted by Helicobacter pylori bacteria that colonize the gastric mucus. 

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). 

Why this elaborate mechanism for getting active digestive enzymes into the gastrointestinal tract Synthesis of the enzymes as inactive precursors protects the exocrine cells from destructive proteolytic attack. The pancreas further protects itself against self-digestion by making a specific inhibitor, a protein called pancreatic trypsin inhibitor (p. 231), that effectively prevents... 

Dissolution or destruction (self-digestion) of cell. Volume... 

The stability of both the wild-type and mutant proteins is expressed as the melting temperature, Tm, which is the temperature at which 50% of the enzyme is denatures during irreversible heat denaturation. To prevent the self-digestion of penicillolysin, heat treatment was carried out at pH 5.0 because the proteolytic activity is substantially reduced at this pH and the active form of the enzyme is stable. For the wild-type enzyme and the three mutants, the thermal stabilities and the 7m changes were assayed at pH 5.0 by measuring the far-UV CD spectrum at 222 nm as a function of temperature. For the wild-type penicillolysin, the Tm... 

As discussed in Section 19.3.5.3, mimicking human digestion for the assessment of Se-bioavailability could be practically regarded as early examples of sequential enzymatic sample preparation applying, for example, proteolytic (pepsin), mixed (pancreatin), and non-proteolytic (amylase) enzymes, and digestive compounds like bile salts for lipid emulsification. On the other hand, the simultaneous use of enzymes was not considered a source of self-digestion problems as this phenomenon is common in the digestive tract. 

Titration curves of trypsin were obtained under a variety of conditions by Duke et al. (1952). The most noteworthy feature is a specific effect of calcium, which displaces the acid part of the titration curve to lower pH, and decreases the total number of groups which are titrated between pH 6 to 9. It is likely that the groups titrated between pH 6 and 9 in the absence of Ca " are a-amino groups, produced by self-digestion of the enzyme. The effect of Ca" " thus appears to result from a complex with the carboxyl groups of the protein, which stabilizes the anionic form of these groups so as to produce the displacement of the acid part of the titration curve. This complex is more resistant to self-digestion than the enzyme alone. 

This will give rise to a set of autolysis peptides from the self-digestion of the enzyme. These autolysis peptides are impossible to eliminate but can be minimized by using the highest quality autolysis-resistant enzyme. Other proteins such as bovine serum albumin (BSA) may be used in the immunopurification of specific proteins. Again, if this is an unavoidable part of the protocol, then the analyst should expect to observe peptide ions derived from these proteins. 

Autoiysis. Self-hydrolysis by tissue-degrading enzymes self-digestion. 

The sections below deal with the biochemistry of the Ca " " ion only insofar as it is involved in catalysis (i.e. enzymatic activity.) They will not deal with Ca " bound to enzymes in which this ion plays a purely passive structural role as in the case of the Zn " " endoproteinase thermolysin which binds a Zn " " ion at the active site and four Ca ions elsewhere which stabilize the structure . Removal of the Ca " " causes this enzyme to autolyze (self-digest) and become inactive, but Ca is not directly involved in the catalytic function. Many examples of Ca acting in a passive structural rule are known. ... 

A reversed micellar system inhibits self-digestion of the enzyme and contamination by micro-organisms. 

Trypsin cleaves proteins at the carboxy terminal side of lysine and arginine residues. Arg-Pro or Lys-Pro sites are trypsin resistant. Also, trypsin only slowly attacks peptide bindings between a basic amino acid (Lys, Arg) and an acidic one (Glu, Asp). The pH optimum of trypsin lies between 8 and 9, and the optimum relation of enzyme to substrate is 1 50 to 100. Ca ions inhibit the self-digestion of trypsin. The trypsin must not contain any chymotrypsin activity (i.e., TPCK must be treated and highly purified). Lysine residues can be protected from trypsin by derivatization (e.g., with citraconic acid), and in reverse, treating the cysteine groups with iodoethylene trifluoroacetamide introduces new trypsin cleaving sites. 

The V8 protease of Staphylococcus aureus (MW 12 kd) is active at a pH between 3.5 and 9.5 and develops maximal activity at pH 4.0 and 7.8. At pH 4.0, the protease partially precipitates. In phosphate buffer, V8 protease cleaves peptide bindings on the carboxyl terminal side of aspartate or glutamate residues. In 50 mM ammonium bicarbonate buffer pH 7.8 or ammonium acetate buffer pH 4.0, on the other hand, the enzyme cleaves only behind glutamate residues. Below 40° C, the protease does not exhibit any self-digestion. Its watery solution can be frozen and thawed without activity loss. Divalent cations or EDTA have no effect on the enzyme activity. The enzyme also still works in 0.5% SDS. Diisopropyl fluorophosphate inhibits V8 protease. 

Mizhushima N, Levine B, Cuervo AM, Klionsky DJ. Autophagy fights disease through cellular self-digestion. Nature 2008 45 1069-1075. 

Figure 3. Identification of a protein by peptide mass fingerprinting. The protein constituents of pig saiiva were separated by SD-PAGE and a protein band was digested with trypsin. The resuitant tryptic peptides were mass-measured using MALDI-ToF mass spectrometry. The peptides in the mass spectrum were either derived from trypsin self-digestion (T) or were derived from the protein in the gel- Database searching with the masses of these peptides led to an unequivocal identification of the protein as SAL (salivary lipocalin). The inset map shows the theoretical tryptic digestion map of this protein, and underneath are the peptides that were observed. In many instances, smaller peptides were visible as partial digestion products.

Levine, B. and Khonsky, D.J. (2004) Development by self-digestion Molecular mechanisms and biological functions of autophagy. Dev. CeU, 6 463-477. 

Although autolysis occurs fairly readily when yeast is stored (and consequently is of importance in influencing beer flavour (see Chapters 16 and 17), the process used to make extracts is accelerated by raising the temperature to 45 C (113°F) in the presence of small amounts of ethyl acetate or chloroform, and often in the presence of zinc salts. After a period of 6-12 hr, the autolysed (self digested) yeast is clarified and concentrated. The autolytic procedure involves the disintegration of the vacuole and the release of lytic enzymes (see Chapter 16). During the process the yeast cells are killed. 

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