Trypsinogen inhibitors

Antitrypsin Blood and other body fluids contain a protein, ai -antitrypsin (ai-AT, currently also called ai-aritiproteinase), that inhibits a number of proteolytic enzymes (also called proteases or proteinases) that hydrolyze and destroy proteins. [Note The inhibitor was originally named oti-antitrypsin because it inhibits the activity of trypsin (a proteolytic enzyme synthesized as trypsinogen... 

Figure B3.1.1 A 15% SDS-polyacrylamide gel stained with Coomassie brilliant blue. Protein samples were assayed for the purification of a proteinase, cathepsin L, from fish muscle according to the method of Seymour et al. (1994). Lane 1, purified cathepsin L after butyl-Sepharose chromatography. Lane 2, cathepsin L complex with a cystatin-like proteinase inhibitor after butyl-Sepharose chromatography. Lane 3, sarcoplasmic fish muscle extract after heat treatment and ammonium sulfate precipitation. Lane 4, sarcoplasmic fish muscle extract. Lanes M, low-molecular-weight standards aprotinin (Mr 6,500), a-lactalbumin (Mr 14,200), trypsin inhibitor (Mr 20,000), trypsinogen (Mr 24,000), carbonic anhydrase (Mr 29,000), gylceraldehyde-3-phosphate dehydrogenase (Mr 36,000), ovalbumin (Mr 45,000), and albumin (Mr 66,000) in order shown from bottom of gel. Lane 1 contains 4 pg protein lanes 2 to 4 each contain 7 pg protein.

Figure B3.1.3 An isoelectric focusing (IEF) gel, pH 3 to 10. Lane 1, 4 pg purified egg white cystatin. Lane M, broad-range pi standards trypsinogen (pi 9.3), lentil lectin-basic band (pi 8.65), lentil lectin-middle band (pi 8.45), lentil lectin-acidic band (pi 8.15), myoglobin-basic band (pi 7.35 visible as a broad band), myoglobin-acidic band (pi 6.85), human carbonic anhydrase B (pi 6.55), bovine carbonic anhydrase (pi 5.85), a-lactoglobulin A (pi 5.20), soybean trypsin inhibitor (pi 4.55), and amyloglucosidase (pi 3.50) in order shown from top of gel. The pi values of the two purified egg white cystatin isomers were determined to be 6.6 (upper band) and 5.8 (lower band). Adapted from Akpinar (1998) with permission from author.

Human pancreatic secretory trypsin inhibitor (hPSTI) can be potentially assayed as an indicator of necrotic complications in AP (Ol). This protein is an inhibitor of trypsinogen, which is produced in acinar cells in the quantity of approximately 2% of the potential content of trypsin in pancreas. Trypsin binds with its inhibitor hPSTI, then with AMG, and only this complex, trypsin-o 2-macroglobulin, is eliminated from plasma (B10). Pezzili (P3) suggests that early attempts to determine the severity of the AP process based on the measurement of hPSTI within 24 hr from the first sensations of pain show a sensitivity of 79%, whereas an increase in CRP concentration has a sensitivity of 29% only (Table 3). 

FIGURE 13 Plot of the logarithm of the retention volume (In VR) versus the concentration of the displacing salt, ammonium sulphate, in the HP-HIC mode with the proteins I, insulin B-chain 2, bovine trypsin inhibitor 3, bovine trypsinogen 4, insulin A-chain 5, ribonuclease 6, sperm whale myoglobin 7, horse heart cytochrome c. Data from Ref. 42. 

Because of the large number of proteases it inhibits, WT (wild type) eco has become useful as a biochemical tool. As a selective inhibitor of serine proteases, eco has been used in cell culture assays to probe enzymatic activity as well as to titrate the percentage of catalytically active molecules of a previously uncharacterized serine protease [8]. Immobilized eco has been used for the direct purification of trypsinogen [9]. Finally, eco selectively identified a novel protease implicated in prostate cancer, membrane type serine protease I (MT-SPl) [10]. 

Soybean trypsin inhibitor 4.6 Trypsinogen (bovine pancreas) 9.3... 

Using trypsinogen as biocatalyst under similar conditions in the dropping experiment, after 400 min analytical HPLC showed 21% of 26 and 79% of 25. The conversion of the acyl donor ester was only 80%. Preparative RP-HPLC provided 26 yield Img (7%). The determined amino acid composition corresponded with the calculated values. In the appropriate batch experiment the analytical yield was 17% of 26 (95% conversion of 24), whereas the application of trypsinogen inhibited by basic pancreatic trypsin inhibitor provided only 8% of 26 after 24 h at 48% conversion of the acyl donor ester 24. Using trypsin instead of the zymogen, after approximately 20 min, more than 50% of 26 (after Imin, 20% product formation at 100% conversion of 24) and 25 were digested. 

Hereditary pancreatitis cationic trypsinogen gene mutations, trypsin inhibitor gene mutations Pancreatic insufficiency syndrome... 

Zymogen (e.g., trypsinogen and chymotrypsinogen) synthesis, secretion, transport, and activation and the rate of inactivation of the active enzyme by inhibitors may all be considered means of enzyme regulation. 

Pancreatic juice contains the proenzymes trypsinogen, chymotrypsinogen, procarboxypeptidases, and proelas-tase. All are activated by trypsin in the intestinal lumen. Enteropeptidase located in the brush border of the jejunal mucosa converts trypsinogen to trypsin. A trypsin inhibitor in pancreatic juice protects against indiscriminate autodigestion from intraductal activation of trypsinogen. Other enzymes of pancreatic juice and their substrates are listed below. 

Bode, W, P. Schwager, R. Huber, The transition of bovine trypsinogen to a trypsin-like state upon strong ligand binding. The refined crystal structures of the bovine trypsinogen— pancreatic trypsin inhibitor complex and of its ternary complex with Ile-Val at 1.9 A resolution. J Mol Biol 1978, 118, 99-112. 

The stability of the zymogens is rather fragile a very small amount of active trypsin can activate trypsinogen to create more trypsin and generate an autocatalytic reaction. Trypsin also activates the other zymogens. The normal pancreas protects itself from this catastrophe by a trypsin inhibitor, a small polypep-... 

The pancreas synthesizes and stores the zymogens in secretory granules. The pancreas also synthesizes a secretory trypsin inhibitor. The need for the inhibitor is to block any trypsin activity that may occur from accidental trypsinogen activation. If the inhibitor were not present, trypsinogen activation would lead to the activation of all of the zymogens in the pancreas, which would lead to the digestion of intracellular pancreatic proteins. Such episodes can lead to pancreatitis. 

Marquart M, J Walter, J Deisenhofer, W Bode and R Huber 1983. The Geometry of the Reachve Site and of the Peptide Groups m Trypsm, Trypsinogen and its Complexes with Inhibitors. Acta Crystallographica B39 480-490. 

Ans. The pancreas secretes an array of proteolytic zymogens which must be activated in the small intestine by trypsin, itself secreted as a pancreatic zymogen, trypsinogen. It is pKJSsible for this zymogen to be prematurely activated to trypsin within the pancreas. However, the pancreas also secretes a specific trypsin inhibitor which binds strongly to and inactivates any trypsin prematurely activated. 

Marquart, M., Walter, J., Deisenhofer, J., Bode, W, and Huher, R. (1983). The geometry of the reactive site and of the peptide groups in trypsin, trypsinogen and its complexes with inhibitors. Acta Crystalhgr. Sect. B. 39, 480- 90. 

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