Proteolytic attack

A small number of proteins, and again insulin is an example, are synthesized as pro-proteins with an additional amino acid sequence which dictates the final three-dimensional structure. In the case of proinsulin, proteolytic attack cleaves out a stretch of 35 amino acids in the middle of the molecule to generate insulin. The peptide that is removed is known as the C chain. The other chains, A and B, remain crosslinked and thus locked in a stable tertiary stiucture by the disulphide bridges formed when the molecule originally folded as proinsulin. Bacteria have no mechanism for specifically cutting out the folding sequences from pro-hormones and the way of solving this problem is described in a later section. 

Cervera and Levine [81] studied the mechanism of oxidative modification of glutamine synthetase from Escherichia coli. It was found that active oxygen species initially caused inactivation of the enzyme and generated a more hydrophilic protein, which still was not a substrate for the protease. Continuous action of oxygen species resulted in the formation of oxidized protein subjected to the proteolytic attack of protease. 

Proteins differ greatly in their intrinsic susceptibility to proteolytic attack. Resistance to proteolysis seems to be dependent upon higher levels of protein structure (i.e. secondary and tertiary structure), as tight packing often shields susceptible peptide bonds from attack. Denaturation thus renders proteins very susceptible to proteolytic degradation. 

Bacteria represent a promising source for the production of industrial enzymes. Bacterial cellulases are an especialfy interesting case in point. Many thermophilic bacterial species produce cellulases that are stable and active at high temperature, resistant to proteolytic attack, and stable to mechanical and chemical denaturation. However, cellulase productivities in bacteria are notoriously low compared to other microbial sources. In this paper bacterial enzyme production systems will be discussed with a focus on comparisons of the productivities of known bacterial cellulase producers. In an attempt to draw conclusions concerning the regulation of cellulase synthesis in bacterial systems, a tentative model for regulation in Acidothennus cellulofyticus has been developed. 

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... 

The insensitivity of so many of the photophysical and photochemical properties to proteolytic degradation down to about half of the original molecular weight is remarkable. It is particularly noteworthy when the location of the bilatriene chromophore near the N-terminus—one of the sites of preferential endogenous proteolytic attack—is taken into account and also the fact that the photochromic behavior of chromopeptides obtained by further protein degradation progressively deteriorates and is eventually lost [8b],... 

The Use of Nonproteolytic Enzymes Prior to Se Speciation It goes without saying that food samples contain components other than proteins. This fact can be related to the observation that some sample preparation attempts based on the sole use of proteolytic enzymes have failed, for example, protease XIV could not extract more than 8 percent of Se from Se-enriched lactic acid bacteria with a chromatographic recovery of 27 percent [77]. There are two possible explanations for this phenomenon (1) formerly unidentified and nonprotein-bound Se species might be present in the samples under test, which cannot be released by proteolytic enzymes from the matrix constituents under the experimental conditions adopted (2) the unextracted Se species are protein-bound, but the proteolytic attack is hampered by a matrix constituent, hindering any enzymatic access. 

The direct acylation and methylation of the free 01-NH2 groups of proteins have been proposed to be useful in providing resistance toward proteolytic attacks. Although the basis for this explanation is not always readily apparent from the known specificities of proteases, it may be valid in some cases. Thus, the acetylated N-terminus of a-crystallin, the major protein found in eye lens (17), is presumably important for the protein to survive in an environment rich in leucine aminopeptidase. On the other hand, it is difficult to rationalize that the acetylated N-terminus of bovine pancreatic a-amylase is in any way responsible for the fact that the enzyme is exceedingly stable against tryptic and chymotryptic digestion (18). The function of the acetylation is, in this case, as obscure as is the basis on which a-amylase is selected for acetylation among the many non-acetylated companion pancreatic proteins. 

Effect of Carbohydrate on Enzyme-Substrate Interaction. Since many of the enzymes we have conjugated with dextran have macromolecules as their natural substrates, we recognized that the conjugation process might result in unfavorable steric interactions that would impair the ability of the enzymes to interact with such substrates, in the same way that attached carbohydrate affects the susceptibility of the conjugated enzymes to proteolytic attack. We have therefore investigated the effect of carbohydrate on the interaction of conjugated enzymes with substrate. 

Oxidative modification of proteins renders them more susceptible to proteolytic attack and enzymic hydrolysis (Wolff et al., 1986 Davies, 1987). Hence, if generation of active oxygen species occurs significantly in vivo, one consequence may be accelerated hydrolysis of damaged proteins. Therefore, radical generation at inappropriate sites may lead to destruction of the protein and pathological tissue degradation. 

In contrast to OmpX the long external loops of OmpA are highly mobile and, on the whole, not visible in the respective electron density map. In vivo, the mobile loops are rather resistant to proteolytic attack, presumably because they bind to the surrounding lipopolysac-charides. Obviously, the mobile loops fulfill essential functions in bacterial life (Morona et al., 1984). They are also known as docking points for bacteriophages. 

Fig. 6.2. Schematic representation of an Ig molecule (IgG). The heavy chains are shaded, and both the light and the heavy chains are shown as strings of domains. The hinge region (hr) is most exposed to proteolytic attack. Depending on protease (and origin of Ig), Fab or F(ab )2 antigen-binding fragments are generated. F(ab )2 can be further reduced.

SC has at least two important functions (i) it transports the secretory IgA (sIgA) through the epithelium to the lumen, and (ii) it confers a greater resistance of the sIgA in the mucous secretions or colostrum against proteolytic attack. Sometimes, IgM with SC is found in patients with an IgA deficiency. 

In contrast to the cellular environment, where enzymatic degradation of proteins is highly controlled, extracellular proteases are the cause of uncontrolled protein degradation. The result of this proteolytic attack may vary from complete hydrolysis, single breaks within the peptide chain, or loss of a few N- or C-terminal amino acid residues. Besides losing the product, presence of truncated forms may seriously challenge the purification design. 

Proteins are probably more resistant toward proteolytic attacks in their native state and stabilizing factors (e.g., co-factor, correct parameter interval, co-solvent) are always considered optimized. Use of protein inhibitors is not recommended for safety reasons. The primary mechanism of proteolysis is the enzymatic hydrolysis of the peptide bond. The indicators of this reaction taking place in the system include loss of product or poor yield, lack of expected activity, changes in specific activity, change in MW, high background staining in ID SDS electrophoresis, smeared bands, and many lower MW bands of poor resolution, disappearance of bands, and discrepancies in MW. The preventive actions taken to prevent proteolysis are listed in Table 5. 

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