Trypsin-link activity

Open-channel titania and alumina-based PDMS microchip Trypsin Covalent linking Covalent immobilization of trypsin using activation reagent l-ethyl-3-(3-dimethyl aniinopropyl)carbodiimide (EDC)/N-hydroxysuccinimide (NHS) Fast flow rate of 3.5 pl/min enabled protein digestion for 5 s Innovative feature performing on-hne protein analysis by embedded stainless steel tubing electrode [78]... 

An isolated flagellum will continue to bend actively, indicating that this function is linked to its intrinsic structure. Treatment of cilia from the protozoan Tetra-hymena with the proteolytic enzyme trypsin selectively dissolves the nexin links and radial spokes but leaves unaffected the microtubules and dynein arms. If such a preparation is treated with a small amount of ATP, the loosened microtubule doublets slide against each other and through longitudinal overlap, extend for a distance that is up to nine times the original length of the cilium (Warner and Mitchell, 1981). 

These proteolytic enzymes are all endopeptidases, which hydrolyse links in the middle of polypeptide chains. The products of the action of these proteolytic enzymes are a series of peptides of various sizes. These are degraded further by the action of several peptidases (exopeptidases) that remove terminal amino acids. Carboxypeptidases hydrolyse amino acids sequentially from the carboxyl end of peptides. They are secreted by the pancreas in proenzyme form and are each activated by the hydrolysis of one peptide bond, catalysed by trypsin. Aminopeptidases, which are secreted by the absorptive cells of the small intestine, hydrolyse amino acids sequentially from the amino end of peptides. In addition, dipeptidases, which are structurally associated with the glycocalyx of the entero-cytes, hydrolyse dipeptides into their component amino acids. 

Plasmin is a serine proteinase (inhibited by diisopropylfluorophosphate, phenylmethyl sulphonyl fluoride and trypsin inhibitor) with a high specificity for peptide bonds to which lysine or arginine supplies the carboxyl group. Its molecular weight is about 81 Da and its structure contains five intramolecular disulphide-linked loops (kringles) which are essential for its activity. 

Schematic diagrams of the amino acid sequences of chymotrypsin, trypsin, and elastase. Each circle represents one amino acid. Amino acid residues that are identical in all three proteins are in solid color. The three proteins are of different lengths but have been aligned to maximize the correspondence of the amino acid sequences. All of the sequences are numbered according to the sequence in chymotrypsin. Long connections between nonadjacent residues represent disulfide bonds. Locations of the catalytically important histidine, aspartate, and serine residues are marked. The links that are cleaved to transform the inactive zymogens to the active enzymes are indicated by parenthesis marks. After chymotrypsinogen is cut between residues 15 and 16 by trypsin and is thus transformed into an active protease, it proceeds to digest itself at the additional sites that are indicated these secondary cuts have only minor effects on the enzymes s catalytic activity. (Illustration copyright by Irving Geis. Reprinted by permission.)...

Fig. 2. Glycoside chains and the Kunitz domains. Amino acid and glycoside sequences for the two Kunitz-binding domains of Bik are shown. C-C across a chain indicates a cross-link. Predicted fragmentation points for Bik due to trypsin exposure are shown by lines with black dots. The aprotinin-matching peptides of each Kunitz domain are shown by a darkened circle. The first peptide of aprotinin is indicated as Aprol. The X-ray structure-predicted touch points between Bik and trypsin are marked. The predicted PI peptide of the active inhibitory site sequence is indicated as well.

Other recent work shows that the lectin Con A, cross-linked with glutaral-dehyde, binds to DEAE-cellulose, irreversibly. The bound lectin possesses adequate biological activity with respect to binding to glycoprotein enzymes (35). Trypsin modified by pyromellitic anhydride binds much better to DEAE-cellulose as compared to native enzyme (M. N. Gupta, unpublished results). 

In recent years a number of authors have reported the specific enzymatic cleavage of the cross-links in collagen (Barkin and Oneson, 1961 High-berger, 1961b Kiihn et al., 1961 Nishihara, 1962 Schmitt, 1963 Kiihn et al., 1963a Rubin et al., 1963 Hafter and Hermann, 1963). The enzymes used include pepsin, ficin, trypsin, and unpurified pancreas extracts of these pepsin is the most effective. As pepsin has no esterase or other nonproteolytic activity it appears that the cross-links include, even if they are not entirely, sections of peptide chains. 

Photostimulation of the catalytic activities of bio-imprinted enzymes was studied by Willner and co-workers [25]. The goal of this study was to control the enzymatic activity with light. They linked a photoactive group such as nitropyran to a-chymo-trypsin. The resulting conjugated enzyme was allowed to interact with A -acetyl-L-phenylalanine (as template) in an an aqueous medium. After precipitating the enzyme-template complex, the template was carefully eluted from the enzyme. The catalytic activities of the non-imprinted and imprinted photoactive enzymes were... 

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