Cleavage by trypsin

Cleavage by Trypsin cuts C-term side of KP unless next residue is P... 

The high thermostability of natural thermophilic enzymes is usually accompanied by increased resistance to other forms of denaturation, such as cleavage by proteases and chemical denaturation by guanidine hydrochloride or urea. A similar trend is seen in the evolved thermostable esterases. Thermostable 8G8 is more resistant than wild type to cleavage by trypsin and to denaturation by guanidine hydrochloride (Gershenson et al, 2000). 

Plasmin Hydrolysis of /3-Casein. Studies on the susceptibility of partially methylated /3-casein to cleavage by trypsin-like enzymes were carried out using the enzyme porcine plasmin. In preliminary investigations, we confirmed that the y-caseins produced by plasmin hydrolysis of native /3-casein were identical with those occurring naturally (28). These are designated yi-, y2-, and y3-casein according to the nomenclature recommendations of Whitney et al. (29) and correspond to residues 29-109, 106-209, and 198-209 of -casein. Presumably the proteose peptone products of plasmin hydrolysis are identical with their natural counterparts, but the latter were not available for comparison. 

The opposite approach, namely, the creation of additional lysine-like sites for cleavage by trypsin has been used in the conversion of cysteine bonds to /S-( -aminoethyl)-cysteine residues by reaction of the thiol groups with jS-bromoethylamine (Lindley, 1959). Enzymatic degradation of such modified proteins, e.g., reduced and /3-aminoethylated insulin, by the ac-... 

The introduction of glycyl residues into proteins with the Leuchs anhydrides of glycine makes peptide bonds next to serine susceptible to cleavage by trypsin (Shalitin, 1961). 

Figure 4.24. Cleavage by Trypsin. Trypsin hydrolyzes polypeptides on the carboxyl side of arginine and lysine residues.

The potassium channel and the sodium channel undergo inactivation within milliseconds of channel opening (Figure 13.28). A first clue to the mechanism of inactivation came from exposing the cytoplasmic side of either channel to trypsin cleavage by trypsin produced a trimmed channel that stayed persistently open after depolarization. A second clue was the finding that alternatively spliced variants of the potassium channel have markedly different inactivation kinetics these variants differed from one another only near the amino terminus, which is on the cytoplasmic side of the channel. 

Since the reagent readily reacts with amino groups at the relatively high concentrations required for complete derivatization of the protein, protection of the -amino group is essential if specific cleavage by trypsin at lysine residues is desired. A possible method of protection prior to derivatization with compound I would involve citraconylation of the lysine residues prior to reaction. 

Figure 2. Three-dimensional model of the peptide 1 cleavage by trypsin based on the X-ray structure of the protein.

Nielsen, M. A., Resende, M., Alifrangis, M., Turner, L., Hviid, L., Theander, T. G., and Salanti, A. (2007). Plasmodium falciparum VAR2CSA expressed during pregnancy-associated malaria is partially resistant to proteolytic cleavage by trypsin. Exp. Parasitol. 117,1-8. 

Similarly, chymotrypsinogen is activated by cleavage by trypsin after Arg-15. The active chymotrypsin (called p-chymotrypsin) can further digest other p-chymotrypsin molecules to release two small peptides and give a-chymotrypsin which is also active. 

The exocrine pancreas secretes phospholipase A2 in an inactive zymogen form, prophospholipase A2. The enzyme is activated in the intestinal lumen by proteolytic cleavage by trypsin. Pancreatic lipase, however, is secreted in its active form, and only needs to bind colipase and substrate to be active. 

Note Trypsin is a serine protease that specifically cleaves at the C-terminal side of arginine (R) and lysine (K). Restrictions to the specificity of trypsin occur when prohne (P) is at the C-terminal side of R or K these peptide bonds are almost completely resistant to cleavage by trypsin. 

Cleavage by trypsin is after Arg and yields a dipeptide therefore, this must be the peptide - Leu-Gly-Ala-Ser-Arg-Phe-Glu... 

Cleavage by Trypsin cut C-term side of KP unless next residue is P Itunt er of R>ass values searched 27 Nunber aC maaa Values aatetaad 21 Sequence coverage ... 

Fig. 10.5. Three-dimensional structure of elastase (according to Sawyer et a/., 1978) indicating the two domains. An arrow indicates the cleavage by trypsin (courtesy of H. C. Watson).

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