Resistance to tryptic digestion

As previously noted, rt-PA is resistant to tryptic digestion if the disulfide linkages are left intact. Hence, to date, no complete direct assignment of these linkages have been made. Another difference which exists in the rt-PA molecule is the presence of an odd number of cysteine residues resulting in at least one free sulfhydryl group in the protein. 

Arginine residues modified by nitromalondialdehyde are resistant to tryptic digestion. However, reduction of the derivative by sodium borohydride yields tetrahydropyrimidyl compounds which are susceptible to hydrolysis by trypsin. Modification by nitromalondialdehyde thereby permits the restriction of tryptic digestion to lysine... 

Ovomucoid is quite resistant to tryptic digestion. In fact, each of the three electrophoretic components exhibits pronounced antitryptic activity (97). 

In the raw state, mature soybeans and many other plant foodstuffs contain protease Inhibitors that diminish the proteolytic activities of trypsin and chymotrypsln in the Intestinal tract, cause pancreatic hypertrophy and suppress growth. Trypsin Inhibitors (TI) account for about 40% of the pancreatic hypertrophic effect and growth-inhibitory capacity of raw soy proteins. The resistance of the raw undenatured protein to tryptic digestion accounts for the remaining 60%. The practical significance of residual TI activity In heat-processed soy protein products and the biochemical effects of other protease Inhibitors have been reviewed (40). 

Commercially available BSA has been found to vary in its susceptibility to tryptic digestion (King and Spencer, 1970 Habeeb, 1977u). Some batches were resistant to trypsin, and the susceptibility to tryptic digestion paralleled the availability of disulfide bonds to reduction in the... 

Additional support for the occurrence of a continuous reorganization process of the membrane components throughout the development can be found in results of experiments in which the susceptibility of various membrane peptides to tryptic digestion, carried out under controlled conditions from the outer surface of the thylakoid, was tested at different stages of the greening process. As shown in Fig. 26, peptides which were exposed to tryptic attack in membrane remnants of dark-grown cells, became protected, whereas others which were susceptible became resistant to trypsinization. 

A significant challenge for neuroscientists in studying the membranous synaptosomal proteome rests squarely in the analysis of its constituent hydrophobic and membrane-bound proteins. These include receptors, transporters, ion channels, and the molecular machinery for synaptic vesicle cycling. Hydrophobic and membrane-bound proteins are poorly resolved by traditional lEF gel technology. Similarly, they tend to resist in-gel tryptic digestion, leading to poor rates of protein identification by PMF (van Montfort et al. 2002). 

When Yamakawa (Y2) studied the gastric juice proteins in regard to resistance to alkali, formalin, and tryptic digestion, he found that most were more resistant to these agents than serum proteins. He opposed, therefore, the concept that proteins of gastric juice are derived from serum in which, as we now know, he was only partly correct. 

Gottschlich et al. [134] developed a microfluidic system that integrated enzymatic reactions, electrophoretic separation of the reactants from the products, and postseparation labeling of the proteins and peptides prior to fluorescence detection (see Fig. 12). Tryptic digestion of oxidized insulin p-chain was performed in 15 min under stopped flow conditions in a heated channel serving as the reactor, and the separation was completed in 60 s. Localized thermal control of the reaction channel was achieved using a resistive heating element. The separated reaction products were then labeled with naphthalene-2,3-dicarboxaldehyde (NDA) and detected by fluorescence detection. 

Finally, Fleutbaaij et al. (2014) noticed that analysis of as little as 10 ng of a tryptic digest results in the iderttification of 300-500 nniqne peptides from 100 to 200 proteins. Therefore, it is obvions that the same analysis can reveal not only P-lactamase resistance but also the identity of bacterial species harboring the resistance phenotype. 

Another study on P. aeruginosa (Peng et al. 2005) examined the sarcosine-in-soluble outer membrane fraction upon treatment with ampidllin, kanamycin, and tetracycline to identify proteins related to the respective antibiotic resistances. The authors found 11 difierential proteins, which were excised from the 2D gel and identified by MALDl-TOF MS after in-gel tryptic digestion of the excised spots. Apart from some known antibiotic resistance proteins, Peng et al. discovered some new proteins and thereby novel potential antibiotic targets. 

The same technique of in-gel tryptic digestion and subsequent identification by MALDl-TOF MS was apphed by Dupont and cowoikers in Acinetobacter bau-mannii, an opportunistic bacillus comprising increasing numbers of resistant strains (Dupont et al. 2005). They compared the outer membrane of different strains and found two differentially expressed proteins, one of which was identified as belonging to the OprD family. 

In conclusion, tryptic digestion of proteins and analysis of the resulting peptides by MALDI-TOF MS or LC-MS have proven to be a potent tool to elucidate mechanisms underlying bacterial resistance to antibiotics. Either proteins of interest or protein fractiorts such as outer membrane proteirrs can be analyzed, or the whole proteome of antibiotic-susceptible and -resistant strairrs can be compared. The proteins of interest or the differentially expressed proteirrs can be digested to... 

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. 

A brief discussion of the chemical reactivity of the products of these enzymes is central to our proposed use of these enz)nnes as antinutritive bases of resistance. Polyphenol oxidase (PPO) and peroxidase (POD) oxidize phenolics to quinones, which are strong electrophiles that alkylate nucleophilic functional groups of protein, peptides, and amino acids (e.g., -SH, -NHof -HN-, and -OH)(Figure 1)(53,63-65). This alkylation renders the derivatized amino acids nutritionally inert, often reduces the digestibility of protein by tryptic and chymotryptic enzymes, and furthermore can lead to loss of nutritional value of protein via polymerization and subsequent denaturation and precipitation (63,66-69). POD is also capable of decarboxylating and deaminating free and bound amino acids to aldehydes (e.g., lysine, valine, phenylalanine. 

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